Structural differences within the loop E motif imply alternative mechanisms of viroid processing

Authors: Owens, Robert; BAUMSTARK, T - UNIV OF SCIENCES PHILA PA

Submitted to: RNA
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2007
Publication Date: 6/1/2007
Citation: Owens, R.A., Baumstark, T. 2007. Structural differences within the loop E motif imply alternative mechanisms of viroid processing. RNA. 13:824-834.

Interpretive Summary: Viroids are the smallest known agents of infectious disease – small, circular RNA molecules that lack the coat protein characteristic of most conventional viruses yet are able to multiply and cause disease in susceptible host plants. Small sequence changes in a small region of Potato spindle tuber viroid (PSTVd) known as the “loop E motif “ have been shown to have dramatic effects on symptom expression. We are trying to extend these results to Citrus viroid III (CVd-III), a distantly related viroid that can be used to dwarf citrus trees growing on certain rootstocks and thereby reduce production costs. This manuscript i) compares the structures of the loop E motif in these two viroids and ii) uses this information to gain new insights into the molecular biology of CVd-III replication and interaction with its hosts. The knowledge gained will be used to create more effective viroid dwarfing agents for citrus production, and this report will be of greatest interest to researchers interested in the molecular interactions between viroids or viruses and their plant hosts.

Technical Abstract: Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multi-branched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson-Crick GC pairs results in a significant overall stabilization of the CVd-III loop E motif. Unlike PSTVd, the upper strand of the CVd-III loop E motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5' side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3'-sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5' and 3' termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5' and 3' termini of CVd-III in position for the host ligase.