Cryo-EM Structure of a Begomovirus Geminate Particle.

Tobacco curly shoot virus, a monopartite begomovirus associated with betasatellite, causes serious leaf curl diseases on tomato and tobacco in China. Using single-particle cryo-electron microscopy, we determined the structure of tobacco curly shoot virus (TbCSV) particle at 3.57 Šresolution and confirmed the characteristic geminate architecture with single-strand DNA bound to each coat protein (CP). The CP⁻CP and DNA⁻CP interactions, arranged in a CP⁻DNA⁻CP pattern at the interface, were partially observed. This suggests the genomic DNA plays an important role in forming a stable interface during assembly of the geminate particle.

The 3.3 Å structure of a plant geminivirus using cryo-EM.

Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of Ageratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves. Our map also contains density for ~7 bases of single-stranded DNA bound to each CP, and we show that the interactions between the genome and CPs are different at the interface than in the rest of the capsid. With additional mutagenesis data, this suggests a central role for DNA binding-induced conformational change in directing the assembly of geminate capsids.

Near-Atomic Resolution Structure of a Plant Geminivirus Determined by Electron Cryomicroscopy.

African cassava mosaic virus is a whitefly-transmitted geminivirus which forms unique twin particles of incomplete icosahedra that are joined at five-fold vertices, building an unusual waist. How its 22 capsomers interact within a half-capsid or across the waist is unknown thus far. Using electron cryo-microscopy and image processing, we determined the virion structure with a resolution of 4.2 Å and built an atomic model for its capsid protein. The inter-capsomer contacts mediated by the flexible N termini and loop regions differed within the half-capsids and at the waist, explaining partly the unusual twin structure. The tip of the pentameric capsomer is sealed by a plug formed by a turn region harboring the evolutionary conserved residue Y193. Basic amino acid residues inside the capsid form a positively charged pocket next to the five-fold axis of the capsomer suitable for binding DNA. Within this pocket, density most likely corresponding to DNA was resolved.

Disassembly of African cassava mosaic virus.

The plant-infecting geminiviruses encapsidate their single-stranded DNA genome in characteristic twinned particles that are unique among viruses. These particles are formed by joining two incomplete T=1 icosahedra. African cassava mosaic virions were purified by density-gradient centrifugation from infected Nicotiana benthamiana plants and analysed for their stability with respect to pH changes and heat treatment by using electron microscopy. Negative staining and rotary shadowing revealed stable virions as well as isolated capsomeres between pH 4.0 and 8.5. At pH 9.0 and above, particles disintegrated, whereas they mainly aggregated at a pH below 6.0. Heating the preparations to 55 degrees C and above resulted in the complete loss of any discernible structure. A low proportion (approx. 10 %) of particles ejected their DNA within the pH range of 6.0-8.5. Most virions released their DNA at the top (15.9 %) or the shoulder (71.4 %) of the twin particles and only 12.7 % at the waist. Compared with the expected numbers of pentameric capsomeres at the top (9 %), the shoulder (45.5 %) or the waist (45.5 %), the results revealed a preferential DNA release from the top and shoulder of the geminate particle.