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...

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The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

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