STRUCTURES OF THE NATIVE AND SWOLLEN FORMS OF COWPEA CHLOROTIC MOTTLEVIRUS DETERMINED BY X-RAY CRYSTALLOGRAPHY AND CRYOELECTRON MICROSCOPY

Background: RNA-protein interactions stabilize many viruses and also t he nucleoprotein cores of enveloped animal viruses (e.g. retroviruses) . The nucleoprotein particles are frequently pleomorphic and generally unstable due to the lack oi strong protein-protein interactions in th eir capsids. Principles governing their structures are unknown because crystals of such nucleoprotein particles that diffract to high resolu tion have not previously been produced. Cowpea chlorotic mottle virion s (CCMV) are typical of particles stabilized by RNA-protein interactio ns and it has been found that crystals that diffract beyond 4.5 Angstr om resolution are difficult to grow. However, we report here the purif ication of CCMV with an exceptionally mild procedure and the growth of crystals that diffract X-rays to 3.2 Angstrom resolution. Results: Th e 3.2 Angstrom X-ray structure of native CCMV, an icosahedral (T=3) RN A plant virus, shows novel quaternary structure interactions based on interwoven carboxyterminal polypeptides that extend from canonical cap sid 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-electr on microscopy and image reconstruction. Modeling of this structure wit h the X-ray coordinates of the native subunits shows that the 29 Angst rom 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: Th e CCMV capsid displays quaternary structural interactions that are uni que compared with previously determined RNA virus structures. The loos ely coupled hexamer and pentamer morphological units readily explain t heir versatile reassembly properties and the pH and metal ion dependen t polymorphism observed in the virions. Association of capsomeres thro ugh inter-penetrating carboxy-terminal portions of the subunit polypep tides has been previously described only for the DNA tumor viruses, SV 40 and polyoma.