Ensemble of Secondary Structures for Encapsidated Satellite Tobacco Mosaic Virus RNA Consistent with Chemical Probing and Crystallography Constraints

Abstract

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold.

Visualizing RNA base-pairing probabilities with RNAbow diagrams

“…Chemical mapping experiments indicate which bases are unpaired, and this information can also be visualized with Difference RNAbows. Here, for the 5 ′ -UTR region of HIV-1, we compare the constrained (Schroeder et al 2011) partition function where lowercase bases are forced to be unpaired (top, red) with the unconstrained partition function (bottom, blue). R-chie package (Lai et al 2012) also uses double arcs to visualize helices predicted from multiple alignments with the Transat program (Wiebe and Meyer 2010).…”

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

“…Our results are consistent with this scenario and may reflect a general strategy exploited by ss-RNA viruses. Schroeder et al have previously emphasized that, when using experimental restraints to develop secondary structure models, there are generally many different models that will be consistent with those restraints (Schroeder et al 2011). This represents an important direction for future research on RNA secondary structures.…”

“…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). Figure 1A shows our SHAPE-directed secondary structure model.…”

“…In part, this is because RNA is a flexible polymer that is unlikely to adopt a single unique 3D-structure at equilibrium. In addition, it has been hypothesized that large RNAs (of order 1000 nt) may assume an ensemble of secondary structures, each containing a different combination of intramolecular base-pairings between nucleotides in both local and distant regions of the primary sequence, resulting in very different branching patterns (Schroeder et al 2011;Gopal et al 2012;Wu et al 2013). Moreover, the secondary structures may well contain kinetically trapped portions that are formed during replication.…”

“…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).…”