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Journal of Virology, January 2009, p. 927-939, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.02060-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection{triangledown} ,{dagger}

Rong Guo,1 Wai Lin,2 Jiuchun Zhang,1 Anne E. Simon,1 and David B. Kushner2*

Department of Cell Biology and Molecular Genetics, University of Maryland—College Park, College Park, Maryland 20742,1 Department of Biology, Dickinson College, Carlisle, Pennsylvania 170132

Received 30 September 2008/ Accepted 1 November 2008

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3'-half shares high sequence similarity with the TCV 3' end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3' end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot {psi}3, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.

* Corresponding author. Mailing address: Department of Biology, Dickinson College, P.O. Box 1773, Carlisle, PA 17013. Phone: (717) 245-1328. Fax: (717) 245-1130. E-mail: kushnerd{at}dickinson.edu

{triangledown} Published ahead of print on 12 November 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, January 2009, p. 927-939, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.02060-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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