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 T=3 icosahedral virus, has a bipartite positive-sense single-stranded RNA genome, with each RNA molecule packaged in a separate particle. In the crystal structure of particles containing RNA2 (~3.6 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
). Because the RNA density in the X-ray map represents an average of the densities at symmetrically equivalent positions, the nucleotide sequence of the visualized RNA remains unknown. STMV, a T=1 icosahedral virus, packages a single strand of RNA containing 1,059 nucleotides. Up to 624 nucleotides, or 59% of the genome, were visualized in the 1.8-Å structure of the particle (9
). In contrast to BPMV, these nucleotides form double-helical segments that structurally approximate A-form RNA. Using predictions for the position of the remaining nucleotides, a model of the packaged genome was proposed in which the RNA is folded into a series of stem-loops that are in intimate contact with the protein shell (9
The invertebrate viruses FHV and PaV are T=3 icosahedral viruses that contain a bipartite RNA genome. The single-stranded RNA1 and RNA2 molecules contain roughly 3,100 and 1,400 nucleotides, respectively, and are packaged into a single virion. The high-resolution X-ray structure of FHV revealed 10-bp fragments of RNA forming A-type duplexes at the icosahedral twofold contacts of the particle (7
). This RNA accounts for approximately 600 ribonucleotides, or 13% of the encapsidated genome. The RNA duplexes observed at equivalent positions in PaV were significantly longer, comprising a stretch of 25 bp (20
). Modeling of this duplex RNA into the density at the 30 twofold contacts gave the impression of a dodecahedral RNA cage that sits directly beneath the protein shell. This cage provides an average view of ~35% of the encapsidated RNA.
The structural data confirmed earlier Raman spectral analyses of other viruses, which had indicated that as many as ~60% of the bases in packaged, single-stranded RNA genomes are involved in hydrogen bonding (12
). Given the fact that large portions of the encapsidated RNA are engaged in secondary structure interactions, the question arises whether these interactions are simply dictated by the primary sequence of the RNA or whether exogenous factors contribute to the final organization of the RNA in the virion. Thermodynamic considerations suggest that unassisted folding of the genome prior to assembly is highly unlikely, because it would give rise to numerous conformations, few of which would have the properties observed in the crystal structures of the particles. It is more likely that the folding process is guided by interaction with a cofactor, presumably the capsid protein.
In the work described here we have begun to address these issues by investigating the RNA structure in native and mutant FHV particles. Although assembly of FHV particles requires RNA, it is not dependent on the presence of viral RNAs 1 and 2. Particles containing heterologous RNAs can be generated using recombinant baculovirus expression vectors (16
). We used electron cryomicroscopy (cryoEM) and image reconstruction to analyze four types of FHV particles that differed in their protein and RNA content. We found that encapsidated viral and heterologous RNAs have virtually identical dodecahedral structures adjacent to the capsid protein, indicating that the FHV coat protein and not the sequence of the nucleic acid controls the organization of this region of the packaged RNA. An N-terminal basic segment of the coat protein known to interact with the packaged genome is not required for this function.