This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Price, B. D.
Right arrow Articles by Ball, L. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Price, B. D.
Right arrow Articles by Ball, L. A.

 Previous Article  |  Next Article 

Journal of Virology, February 2002, p. 1610-1616, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1610-1616.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

DNA-Directed Expression of an Animal Virus RNA for Replication-Dependent Colony Formation in Saccharomyces cerevisiae

B. D. Price,1* P. Ahlquist,2 and L. A. Ball1

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294,1 Howard Hughes Medical Institute and Institute for Molecular Virology, University of Wisconsin—Madison, Madison, Wisconsin 537062

Received 17 August 2001/ Accepted 13 November 2001

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 108 plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

* Corresponding author. Mailing address: Department of Microbiology, University of Alabama--Birmingham, BBRB 373/17, 845 19th St. South, Birmingham, AL 35294-2170. Phone: (205) 934-0454. Fax: (205) 934-1636. E-mail: bdp{at}uab.edu.

Journal of Virology, February 2002, p. 1610-1616, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1610-1616.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Stapleford, K. A., Rapaport, D., Miller, D. J. (2009). Mitochondrion-Enriched Anionic Phospholipids Facilitate Flock House Virus RNA Polymerase Membrane Association. J. Virol. 83: 4498-4507 [Abstract] [Full Text]  
  • Weeks, S. A., Miller, D. J. (2008). The Heat Shock Protein 70 Cochaperone YDJ1 Is Required for Efficient Membrane-Specific Flock House Virus RNA Replication Complex Assembly and Function in Saccharomyces cerevisiae. J. Virol. 82: 2004-2012 [Abstract] [Full Text]  
  • Annamalai, P., Rofail, F., DeMason, D. A., Rao, A. L. N. (2008). Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration. J. Virol. 82: 1484-1495 [Abstract] [Full Text]  
  • Price, B. D., Eckerle, L. D., Ball, L. A., Johnson, K. L. (2005). Nodamura Virus RNA Replication in Saccharomyces cerevisiae: Heterologous Gene Expression Allows Replication-Dependent Colony Formation. J. Virol. 79: 495-502 [Abstract] [Full Text]  
  • Miller, D. J., Schwartz, M. D., Dye, B. T., Ahlquist, P. (2003). Engineered Retargeting of Viral RNA Replication Complexes to an Alternative Intracellular Membrane. J. Virol. 77: 12193-12202 [Abstract] [Full Text]  
  • Dasgupta, R., Cheng, L.-L., Bartholomay, L. C., Christensen, B. M. (2003). Flock house virus replicates and expresses green fluorescent protein in mosquitoes. J. Gen. Virol. 84: 1789-1797 [Abstract] [Full Text]  
  • Miller, D. J., Ahlquist, P. (2002). Flock House Virus RNA Polymerase Is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion. J. Virol. 76: 9856-9867 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»