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Journal of Virology, September 2006, p. 8566-8581, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00837-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication

Xuehua Zhong,¹ Neocles Leontis,² Shuiming Qian,^3,4 Asuka Itaya,¹ Yijun Qi,^1, Kathleen Boris-Lawrie,^3,4,5 and Biao Ding^1,4,5*

Department of Plant Cellular and Molecular Biology and Plant Biotechnology Center, Ohio State University, Columbus, Ohio 43210,¹ Department of Chemistry and Center for Biomolecular Sciences, Bowling Green State University, Bowling Green, Ohio 43404,² Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210,³ Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio 43210,⁴ OSU RNA Consortium, Ohio State University, Columbus, Ohio 43210⁵

Received 23 April 2006/ Accepted 19 June 2006

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.

Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

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