GENOME PACKAGING IN SINGLE-STRANDED RNA VIRUSES
The structure of satellite tobacco mosaic virus (STMV) (2012):
Our model of STMV, developed by graduate student Yingying Zeng, was the first all-atom model ever published for any virus. It included the genomic RNA sequence and incorporated an RNA secondary structure model based on chemical probing, from the laboratory of Susan Schroeder (University of Oklahoma). The model was based on the x-ray crystal structure of the virus, which revealed an icosahedrally averaged view of 30 RNA double helices (about 60% of the genome) from the laboratory of Alex McPherson. Our collaborators on this project were Steve Larson and Alex McPherson (UC Irvine), and Christine Heitsch (Georgia Tech).
The methods were based in part on work described below, done by an earlier grad student, Batsal Devkota.
Modeling the structures of RNA in single-stranded RNA bacteriophages (2009):
Small ssRNA bacteriophages contain genomes of a few thousand nucleotides, packaged tightly inside the protein capsid. It is a great challenge to develop a stereochemically plausible all-atom model for these assemblies, because of uncertainties in the RNA secondary structure, and because the packing is so dense. The latter is a particularly serious obstacle, because there are no automated methods capable of generating model structures without unacceptable steric overlaps, and because there is no room in which to manually manipulate the model.
We devised a method of overcoming the crowding problem, using pariacoto virus (PaV) as the prototype system. The secondary structure of PaV RNA is not known, but the crystal structure revealed the locations of 30 symmetrically placed RNA duplexes, so we created a hypothetical secondary structure that would satisfy the crystallographic observation and invented a sequence to match that structure. We took crystal structure of the capsid and expanded it radially to generate plenty of space for anchoring RNA secondary structure elements to the inside of the capsid and connecting them together. We then built models of the connections between these and gradually shrank the capsid down to the correct diameter, using appropriate restraints to maintain the RNA-protein anchors and pull the whole thing together. This effort was led by Batsal Devkota, a graduate student in the Harvey lab, and involved collaborations with Sébastien Lemieux, Liang Tang, Anette Schneemann and Jack Johnson. The result was the first all-atom model of an RNA virus (Devkota et al., 2009), but we did not use the actual viral RNA sequence, and the RNA secondary structure was not based on data from the RNA itself.