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Journal of Virology, May 2005, p. 5743-5751, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5743-5751.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

Jessica E. Petrillo, Gail Rocheleau, Brenna Kelley-Clarke, and Lee Gehrke*

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

Received 13 September 2004/ Accepted 23 December 2004

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3' untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3' organization model as an alternate model of AMV replication that offers an improved fit to the available data.

* Corresponding author. Mailing address: HST Division, MIT E25-545, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-7608. Fax: (509) 357-7835. E-mail: lee_gehrke{at}hms.harvard.edu.

Journal of Virology, May 2005, p. 5743-5751, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5743-5751.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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