| Summary: | The genomes of viruses must be packaged into a protective protein shell known as the capsid. For simple viruses with a positive-sense, single-stranded RNA genome, this packaging is specific, despite the presence of a large number of competing cellular transcripts. Interactions between the genome and capsid proteins must at least in part determine the selectivity of packaging. and the organization of the packaged RNA. This organization is difficult to visualize as genomic RNAs are large complex molecules that can in principle adopt many different conformations and orientations within the viral capsid. This thesis describes the use of electron microscopy techniques to determine the structure of simple positive-sense RNA icosahedral viruses. The principal aim has been to study the genome organization of two such viruses using methods based on electron cryo-microscopy (cryo-EM). Single-particle electron microscopy and icosahedral 3D reconstruction has been used to probe the RNA structure and organization of the picorna-like Cowpea mosaic virus (CPMV). Sub-nanometer resolution 3D reconstruction, has allowed the secondary structure elements of the viral capsid to be resolved. We show that the CPMV genomic RNA is organized as a dodecahedral caged structure, and identify RNA binding sites on the capsid inner-surface. The binding sites do not appear to enforce a particular bound conformation on the RNA, as has been observed for members of the Nodaviridae. We also image, for the first time, a C-terminal 24 amino acid segment that is implicated in RNA packaging and virus assembly but missing from previous structural studies. We present a pseudo-atomic model for this sequence. Electron cryo-tomography and sub-tomographic averaging has been used to carry out the asymmetric 3D reconstruction of the bacteriophage MS2:F-pili (virus-receptor) complex to - 40 A resolution. The resulting structure, the first such, provides unique insights into the biology of a single-stranded RNA virus. We describe for the first time, the asymmetric distribution of genomic RNA within an asymmetrically averaged virus capsid, confirming previous ideas about RNA packaging. We show that MS2 is not truly icosahedral, as the viral maturation protein appears to replace one of the capsid protein dimers in the protein shell.
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