Background

RNA-protein interactions stabilize many viruses and also the nucleoprotein cores of enveloped animal viruses (e.g. retroviruses). The nucleoprotein particles are frequently pleomorphic and generally unstable due to the lack of strong protein-protein interactions in their capsids. Principles governing their structures are unknown because crystals of such nucleoprotein particles that diffract to high resolution have not previously been produced. Cowpea chlorotic mottle virions (CCMV) are typical of particles stabilized by RNA-protein interactions and it has been found that crystals that diffract beyond 4.5 A resolution are difficult to grow. However, we report here the purification of CCMV with an exceptionally mild procedure and the growth of crystals that diffract X-rays to 3.2 A resolution.

Results

The 3.2 A X-ray structure of native CCMV, an icosahedral (T = 3) RNA plant virus, shows novel quaternary structure interactions based on interwoven carboxyterminal polypeptides that extend from canonical capsid beta-barrel subunits. Additional particle stability is provided by intercapsomere contacts between metal ion mediated carboxyl cages and by protein interactions with regions of ordered RNA. The structure of a metal-free, swollen form of the virus was determined by cryo-electron microscopy and image reconstruction. Modeling of this structure with the X-ray coordinates of the native subunits shows that the 29 A radial expansion is due to electrostatic repulsion at the carboxyl cages and is stopped short of complete disassembly by preservation of interwoven carboxyl termini and protein-RNA contacts.

Conclusions

The CCMV capsid displays quaternary structural interactions that are unique compared with previously determined RNA virus structures. The loosely coupled hexamer and pentamer morphological units readily explain their versatile reassembly properties and the pH and metal ion dependent polymorphism observed in the virions. Association of capsomeres through inter-penetrating carboxy-terminal portions of the subunit polypeptides has been previously described only for the DNA tumor viruses, SV40 and polyoma.

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few ångstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps.