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 Rocheleau, G.
Right arrow Articles by Gehrke, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rocheleau, G.
Right arrow Articles by Gehrke, L.

 Previous Article  |  Next Article 

Journal of Virology, August 2004, p. 8036-8046, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8036-8046.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Gail Rocheleau,{dagger} Jessica Petrillo,{dagger} Laura Guogas, and Lee Gehrke*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 11 December 2003/ Accepted 3 April 2004

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.

* Corresponding author. Mailing address: HST Division, MIT E25-545, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-7608. Fax: (617) 253-3459. E-mail: Lee_Gehrke{at}hms.harvard.edu.

{dagger} These authors contributed equally to the work.

Journal of Virology, August 2004, p. 8036-8046, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8036-8046.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Petrillo, J. E., Rocheleau, G., Kelley-Clarke, B., Gehrke, L. (2005). Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication. J. Virol. 79: 5743-5751 [Abstract] [Full Text]  
  • Guogas, L. M., Laforest, S. M., Gehrke, L. (2005). Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent. J. Virol. 79: 5752-5761 [Abstract] [Full Text]  
  • Guogas, L. M., Filman, D. J., Hogle, J. M., Gehrke, L. (2004). Cofolding Organizes Alfalfa Mosaic Virus RNA and Coat Protein for Replication. Science 306: 2108-2111 [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»