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.
Highlights
Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded, positive-sense RNA genome, 1058 nucleotides in length
Reverse transcription using fluorescently labeled primers gives cDNA fragments whose lengths are determined by locations of the
We first probed the in vitro transcribed STMV RNA in the presence of 250 mM Na+ using the selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) reagent Nmethylisatoic anhydride (NMIA). Under these conditions, one would expect the formation of secondary structure, but not necessarily tertiary structure [15,16,17]
Summary
Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded, positive-sense RNA genome, 1058 nucleotides in length. The RNA that is visible is revealed as 30 double-helical segments, each 9 base pairs in length and closely associated with dimers of coat protein (Figure 1). The helical axes are perpendicular to the icosahedral 2fold axes, forming part of the edges of an icosahedron With this constraint on the structure, Larson and McPherson proposed that the RNA forms a series of stem-loop substructures, with only short-range (local) base pairing. They suggested that coat proteins bind to successive stem-loops as these form upon emerging from the replication complex [5]. The results of atomic force microscopy (AFM) experiments are consistent with this hypothesis [6]
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