Carolina G. Casado, Javier Ortiz, Mavis Agbandje-McKenna

Our cryo-electron microscopy and image reconstruction work on MSV-N geminate particles resulted in the first structure determined for a geminivirus. The structure verified previous claims that the geminate particle is made up of 110 copies of the coat protein (CP). The CPs are arranged in 22 pentameric subunits (capsomers) with quasi-icosahedral symmetry. Each "head" contained 11 capsomers and each capsomer has 5 CPs. An atomic model of the MSV-N CP built into the 25 Å resolution cryo-EM image reconstruction of the MSV virion showed that the CP contains an eight-stranded b-barrel motif, a feature common to the CP structure of most icosahedral viruses. This motif is also present in all T=1 icosahedral ssDNA viruses for which atomic structures are known. The b-barrel motif forming the core MSV capsid structure, with loop regions between the strands formed the surface features, as has been observed for the other ssDNA viruses. Work is ongoing to further characterize the MSV CP using biochemical methods and X-ray crystallography. In addition the MSV-N CP model is aiding the structure-based analysis of the CPs of members of the four genera of the Geminiviridae.

Biohysical methodologies, such as X-ray crystallography and cryo-electron microscopy (Cryo-EM) combined with X-ray crystallography, enable the atomic visualization of interaction interfaces between protein subunits and between proteins and nucleic acids in macromolecular aseemblages, including viruses. These studies have demostrated the requirement for a high degree of fidelity in the interactions between components, within a precise geometric constraint, in the steps that precede and result in the assembly of mature infectious virus particles. They also suggest the possibility for viral control based on assembly disruption. However, the extent to which the available information applies to the unique twined icosahedral virus capsid arrangment of the geminiviruses is unkown. This project aims to fill this dearth in our knowledge base. The model virus to be exploited is the well characterized Nigerian strain of the plant geminivirus maize streak virus (MSV-N). The nature of the MSV coat protein, the geometric components in the MSV viral capsid assembly pathway and the essential protein-protein and protein-DNA interactions that are required for building the unique geminate capsid are examined by biochemical and structural (X-ray crystallography and cryo-EM) methods. These studies should validate the computational models to be developed for probing the driving force behind the MSV geminate capsid assembly.