J. Jared Gossett’s research while affiliated with Georgia Institute of Technology and other places

Some small icosahedral RNA viruses (e.g., MS2) require a specific packaging signal for the formation of virus particles. Others (e.g., cowpea chlorotic mottle virus (CCMV); Pariacoto virus (PaV)) are able to encapsidate a wide variety of RNAs, forming virus-like particles (VLPs) whose structure are only slightly different from that of the wild-type virus. We have determined the structure of the genomic RNA of satellite tobacco mosaic virus (STMV) in an in vitro transcript (see image below). This structure is very different from the structure of STMV RNA probed inside the virus by Schroeder et al. (BJ 101:167 (2011)), which consists of a string of stem-loops connected by single-stranded regions. We have also developed all-atom models for three viruses: PaV (using a hypothetical secondary structure and a non-viral sequence); STMV (using the true sequence and the Schroeder secondary structure); and MS2 (using the true sequence and a hypothetical secondary structure). In this talk, we discuss the implications of these secondary structures and three-dimensional models for the pathways of viral assembly.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

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.

Combined peak area signal after decay correction. The thick black line fitted to the corrected peak area data has a slope of zero, ensuring that intense values in the beginning, middle, and end of the signal are of uniform height. (TIF)

(PDF)

Signal decay correction. The regions of overlapping data from different primers are not on the same scale (top). After scaling all of the primers to one another such that the overlapping regions match up, the resulting signal decays rapidly (middle). After correcting for signal decay, the overlapping regions are in agreement (bottom). (TIF)

Predicted secondary structures for STMV RNA. SHAPE MFE and Subopts #1–9 were predicted using the SHAPE experimental data as constraints. Default MFE was predicted without the SHAPE data. Each secondary structure is shown as an arc diagram, in which the sequence is arranged along a horizontal line and base pairs are shown as arcs connecting the corresponding bases. The structures are listed in order of ascending pseudo-energy values. Pseudo-energy is the calculated free energy that includes the SHAPE pseudo-energy terms. Also shown are the energy values evaluated using the default energy model parameters ignoring SHAPE terms. MLD is the maximum ladder distance. All structures predicted using RNAstructure version 5.3. (TIF)

Quantitative correlation between peak area data in overlapping primer reads. This demonstrates that signal decay in the regions of overlapping data is similar. Pearson’s r-values are shown. (TIF)

In vitro transcribed STMV RNA runs as a single band on a native gel. STMV RNA is run on a 1% agarose gel. No sample was loaded in lanes 2 or 4. Lanes 1 and 3 contain STMV RNA in SHAPE probing buffer without Mg2+ (50 mM HEPES pH 8.0, 200 mM sodium acetate pH 8.0) and lane 5 contains STMV RNA in 100 mM Tris-HCl pH 8.0. All samples were heated to 90°C for 2 min. Samples in lanes 1 and 5 were snap-cooled by chilling on ice, while the one in lane 3 was allowed to slow-cool to room temperature. The samples were then loaded on the gel using 6X native gel loading dye (New England Biolabs) and stained with SYBR Gold nucleic acid gel stain (Invitrogen). Lanes 1, 3 and 5 contain a single band, indicating a single dominant conformation. (TIF)

Primers used to analyze the STMV RNA. (PDF)