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

Abstract

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

Packaged and Free Satellite Tobacco Mosaic Virus (STMV) RNA Genomes Adopt Distinct Conformational States

“…This is a key finding as early models proposed only local stem loop structures, 4,12,14,22 but current models specifically support these long-range interactions. 9-11 We discuss our predicted secondary structures largely in terms of probability arcs, as these best represent the structural variability of some regions and their contrast to the single structures observed in other regions. In order to facilitate conventional visualization of the two conformations, we also constructed traditional minimum free energy secondary structure models from the SHAPE data (Figure 4; Supplemental Figure 1).…”

Sizes of Long RNA Molecules Are Determined by the Branching Patterns of Their Secondary Structures

“…However, there are several notable discrepancies between the predicted and measured sizes. One limitation of our approach is that computational predictions may yield an incorrect structure and hence an MLD that differs from that of the experimentally determined secondary structure, as in the case of STMV RNA (21,55). Such failures of the computational approach are more likely (Table 1) and coloring is according to the class (black, single-stranded precursors of dsRNA viral genomes; red, genomes of ssRNA viruses; blue, cellular mRNAs; green, ribosomal RNA; and cyan, long noncoding RNAs.…”

Recent advances in coarse-grained modeling of virus assembly

“…Experiments and simulations suggest that the physical features of viral RNAs (e.g. charge, and size due to tertiary structure) are optimized for assembly of their capsid [33,48,[51][52][53][54][55]. Secondly, interactions between capsid proteins and specific sequences within the genome called packaging sites (PSs) have been identified for a number of viruses (e.g.…”