In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

Abstract

Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3′ end. Here we use selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3′ end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

“…They are typically a few hundred nucleotides long, with extended secondary structures that have little or no branching (Wang et al 2004;Xu et al 2012). This structure is believed to provide viroid RNAs with resistance to RNA silencing-mediated degradation (Wang et al 2004), and we proposed a similar role for the STMV secondary structure (Athavale et al 2013), since this RNA can also infect plants in the absence of a capsid coat (Mirkov et al 1990;Routh et al 1995;Dodds 1998).…”

“…In addition to the primary sequence that directly codes for the viral proteins, single-stranded (ss)-RNA viruses exploit their secondary and tertiary structures to direct various events during infection. For example, specific secondary structure motifs within the viral genome have been implicated in packaging (Valegård et al 1994;Qu and Morris 1997;Choi et al 2002;Bunka et al 2011), genome replication (Frolov et al 2001;Lindenbach et al 2002;Damgaard et al 2004), the regulation of protein expression (Barends et al 2003), protein folding (Watts et al 2009), conformational switching (Stockley et al 2007), and evading degradation by the host cell (Athavale et al 2013).…”

“…When such data are used to supplement thermodynamic predictions on relatively short RNAs, significantly more reliable secondary structure maps can be produced (Deigan et al 2009). The data can be used to generate an ensemble of structures (Schroeder et al 2011), although they are commonly interpreted in terms of a single structure (Watts et al 2009;Archer et al 2013;Athavale et al 2013;Wu et al 2013).…”

“…We previously incorporated data from one such technique, selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) (Wilkinson et al 2006), to suggest the most likely secondary structure of the STMV genomic RNA generated by in vitro transcription (Athavale et al 2013). Our model was very different from the ensemble of models based on chemical probing in virio and the assumption that only short-range base pairs exist in the STMV genome in the mature virus (Schroeder et al 2011).…”

“…(For a description of the yeast RNA control, see Materials and Methods.) We noted that the highly extended central domain is reminiscent of viroid RNAs (Athavale et al 2013). These are circularized, nonencapsidated, noncoding ssRNA molecules that infect plants.…”