Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Proc Natl Acad Sci U S A. 1996 Sep 3; 93(18): 9465–9470.
doi: 10.1073/pnas.93.18.9465
PMCID: PMC38451
PMID: 8790353

Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae.

B D Price, R R Rueckert, and P Ahlouist
Author information Copyright and License information PMC Disclaimer

Abstract

Here we describe the first instances to our knowledge of animal virus genome replication, and of de novo synthesis of infectious virions by a nonendogenous virus, in the yeast Saccharomyces cerevisiae, whose versatile genetics offers significant advantages for studying viral replication and virus-host interactions. Flock house virus (FHV) is the most extensively studied member of the Nodaviridae family of (+) strand RNA animal viruses. Transfection of yeast with FHV genomic RNA induced viral RNA replication, transcription, and assembly of infectious virions. Genome replication and virus synthesis were robust: all replicating FHV RNA species were readily detected in yeast by Northern blot analysis and yields of virions per cell were similar to those from Drosophila cells. We also describe in vivo expression and maintenance of a selectable yeast marker gene from an engineered FHV RNA derivative dependent on FHV-directed RNA replication. Use of these approaches with FHV and their possible extension to other viruses should facilitate identification and characterization of host factors required for genomic replication, gene expression, and virion assembly.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.8M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

 
icon of scanned page 9465
9465
icon of scanned page 9466
9466
icon of scanned page 9467
9467
icon of scanned page 9468
9468
icon of scanned page 9469
9469
icon of scanned page 9470
9470
 

Images in this article

Fig. 1

Fig. 1
on p.9466

Fig. 3

Fig. 3
on p.9467

Fig. 4

Fig. 4
on p.9468

Fig. 5

Fig. 5
on p.9469

Click on the image to see a larger version.

Selected References

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

  • Janda M, Ahlquist P. RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S. cerevisiae. Cell. 1993 Mar 26;72(6):961–970. [PubMed] [Google Scholar]
  • Quadt R, Ishikawa M, Janda M, Ahlquist P. Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4892–4896. [PMC free article] [PubMed] [Google Scholar]
  • Selling BH, Allison RF, Kaesberg P. Genomic RNA of an insect virus directs synthesis of infectious virions in plants. Proc Natl Acad Sci U S A. 1990 Jan;87(1):434–438. [PMC free article] [PubMed] [Google Scholar]
  • Ball LA, Amann JM, Garrett BK. Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture. J Virol. 1992 Apr;66(4):2326–2334. [PMC free article] [PubMed] [Google Scholar]
  • Selling BH, Rueckert RR. Plaque assay for black beetle virus. J Virol. 1984 Jul;51(1):251–253. [PMC free article] [PubMed] [Google Scholar]
  • Dasmahapatra B, Dasgupta R, Saunders K, Selling B, Gallagher T, Kaesberg P. Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus. Proc Natl Acad Sci U S A. 1986 Jan;83(1):63–66. [PMC free article] [PubMed] [Google Scholar]
  • Ball LA. Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12443–12447. [PMC free article] [PubMed] [Google Scholar]
  • Zhong W, Dasgupta R, Rueckert R. Evidence that the packaging signal for nodaviral RNA2 is a bulged stem-loop. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11146–11150. [PMC free article] [PubMed] [Google Scholar]
  • Ball LA, Li Y. cis-acting requirements for the replication of flock house virus RNA 2. J Virol. 1993 Jun;67(6):3544–3551. [PMC free article] [PubMed] [Google Scholar]
  • Wu SX, Kaesberg P. Synthesis of template-sense, single-strand Flockhouse virus RNA in a cell-free replication system. Virology. 1991 Jul;183(1):392–396. [PubMed] [Google Scholar]
  • Wu SX, Ahlquist P, Kaesberg P. Active complete in vitro replication of nodavirus RNA requires glycerophospholipid. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11136–11140. [PMC free article] [PubMed] [Google Scholar]
  • Scotti PD, Dearing S, Mossop DW. Flock House virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae). Arch Virol. 1983;75(3):181–189. [PubMed] [Google Scholar]
  • Dasmahapatra B, Dasgupta R, Ghosh A, Kaesberg P. Structure of the black beetle virus genome and its functional implications. J Mol Biol. 1985 Mar 20;182(2):183–189. [PMC free article] [PubMed] [Google Scholar]
  • Gallagher TM, Friesen PD, Rueckert RR. Autonomous replication and expression of RNA 1 from black beetle virus. J Virol. 1983 May;46(2):481–489. [PMC free article] [PubMed] [Google Scholar]
  • Friesen PD, Rueckert RR. Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2. J Virol. 1981 Mar;37(3):876–886. [PMC free article] [PubMed] [Google Scholar]
  • Ball LA. Requirements for the self-directed replication of flock house virus RNA 1. J Virol. 1995 Feb;69(2):720–727. [PMC free article] [PubMed] [Google Scholar]
  • Guarino LA, Ghosh A, Dasmahapatra B, Dasgupta R, Kaesberg P. Sequence of the black beetle virus subgenomic RNA and its location in the viral genome. Virology. 1984 Nov;139(1):199–203. [PubMed] [Google Scholar]
  • Friesen PD, Rueckert RR. Black beetle virus: messenger for protein B is a subgenomic viral RNA. J Virol. 1982 Jun;42(3):986–995. [PMC free article] [PubMed] [Google Scholar]
  • Dasgupta R, Sgro JY. Nucleotide sequences of three Nodavirus RNA2's: the messengers for their coat protein precursors. Nucleic Acids Res. 1989 Sep 25;17(18):7525–7526. [PMC free article] [PubMed] [Google Scholar]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed] [Google Scholar]
  • Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. [PubMed] [Google Scholar]
  • Zhong W, Rueckert RR. Flock house virus: down-regulation of subgenomic RNA3 synthesis does not involve coat protein and is targeted to synthesis of its positive strand. J Virol. 1993 May;67(5):2716–2722. [PMC free article] [PubMed] [Google Scholar]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed] [Google Scholar]
  • Russell PJ, Hambidge SJ, Kirkegaard K. Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae. Nucleic Acids Res. 1991 Sep 25;19(18):4949–4953. [PMC free article] [PubMed] [Google Scholar]
  • Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. [PubMed] [Google Scholar]
  • Schneemann A, Zhong W, Gallagher TM, Rueckert RR. Maturation cleavage required for infectivity of a nodavirus. J Virol. 1992 Nov;66(11):6728–6734. [PMC free article] [PubMed] [Google Scholar]
  • Benton BM, Eng WK, Dunn JJ, Studier FW, Sternglanz R, Fisher PA. Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. Mol Cell Biol. 1990 Jan;10(1):353–360. [PMC free article] [PubMed] [Google Scholar]
  • Rose M, Winston F. Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene. Mol Gen Genet. 1984;193(3):557–560. [PubMed] [Google Scholar]
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
-