Nodavirus Coat Protein Imposes Dodecahedral RNA Structure Independent of Nucleotide Sequence and Length

Abstract

The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T‫3؍‬ symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage.High-resolution X-ray analysis of icosahedral viruses has provided detailed insights into the organization of the viral capsid and the structure of the individual coat protein subunits. The encapsidated nucleic acid, on the other hand, is rarely visualized, in part because it does not conform to the icosahedral symmetry of the virus particle. There are exceptions to this, however, as exemplified by the plant viruses bean pod mottle virus (BPMV) (3) and satellite tobacco mosaic virus (STMV) (10) and the invertebrate nodaviruses Pariacoto virus (PaV) (20) and Flock house virus (FHV) (7). In these viruses, regions of the encapsidated single-stranded RNA genome interact with coat protein subunits at symmetrically equivalent positions, and an average structure of these ordered regions has been visualized at high resolution.BPMV, a Tϭ3 icosahedral virus, has a bipartite positivesense single-stranded RNA genome, with each RNA molecule packaged in a separate particle. In the crystal structure of particles containing RNA2 (ϳ3.6 kb), seven well-ordered ribonucleotides were visible near the icosahedral threefold axes of the virion (3). In addition, a total of 660 ribonucleotides corresponding to almost 20% of the packaged RNA could be modeled into the electron density. The overall structure of the ordered RNA is a single-stranded helix, which approximates that found for one strand of an A-type RNA duplex (3, 4). Because the RNA density in the X-ray map represents an average of the densities at symmetrically equivalent positions, the nucleot...

Electrostatics of capsid-induced viral RNA organization

“…The complexation would not consist of continuous stretches of RNA, but rather would occur in the piecemeal fashion that we have just proposed for ss-RNA segments. 40,41 A similar mechanism has been proposed by Tihova et al,32 who suggested that base pairing between RNA segments in free solution is reversible and that there are a sufficient number of secondary structures of RNA from which to select one that is particularly wellsuited to formation of a dodecahedral cage.…”

“…Tihova et al 32 reported two distinct peaks in the radial density distribution of RNA in flock house virus, a close relative of PaV. This bimodal radial density distribution was also found for a viruslike construct encapsidating nonviral RNA.…”

“…The composite density field was visualized as an isosurface-value plot using the OPENDX data visualization package. We considered two cases corresponding to the experimental work of Tihova et al: 32 one where the inward-pointing and flexible N-terminal tails were present, as in the full PaV, and the case where the N-terminal tails were absent, as in the mutant PaVlike particles. The polyelectrolyte-density isosurface images shown in Fig.…”

“…The image reconstruction of microscopy data showed that RNA folding in the dodecahedral RNA cage occurred independent of the specific sequence of RNA. In addition, we note that in cases where x-ray diffraction and cryo-TEM have demonstrated symmetrical ordering in ss-RNA viruses, the ordered parts of the viral genome are associated with specific regions along the inner capsid surface [32][33][34][35][36] perhaps containing an excess of fixed positive charges. Combining these experimental facts and our previous theoretical work demonstrating the dominance of electrostatic forces in RNA virus assembly, we conjecture that the Pariacoto capsid itself provides a nonsequence-specific opportunity for the templated assembly of negatively charged RNA and that the observed dodecahedral structure of the RNA is "preprogrammed" by the capsid independently of the details of the secondary structure of the RNA from which it is formed.…”

Electrostatic interactions in biological DNA-related systems

“…[407][408][409] Many ss-RNA viruses compact their genomes using strongly basic flexible protein arms on the inner side of capsid proteins that provoke adsorption of flexible ss-RNA genome. [410][411][412][413] All these facts underline the importance of ES forces for DNA/RNA packaging and capsid self-assembly, producing a growing number of theoretical studies on this hot topic in the last years. [414][415][416][417][418][419] A number of theoretical [420][421][422] and computational [423][424][425][426] models exist for the capsid self-assembly.…”