Includes bibliographical references. / Maize streak virus (MSV), circular single stranded DNA (ssDNA) virus (~2.7kb), is the causative agent of Maize streak disease, and is a devastating pathogen that causes severe crop losses to subsistence farmers in sub-Saharan Africa. MSV is transmitted by the leafhopper Cicadulina mbila, and is the type member of the Mastrevirus genus (family Geminiviridae). MSV shares a unique twinned icosahedral ("geminate") virion architecture (22 x 38 nm) with all other family members. Geminate particles consist of 110 coat protein (CP) subunits that assemble onto a circular single-stranded DNA (ssDNA) genome. Each T= I unit is an incomplete icosahedron assembled from 55 CPs. The structures of MSV and African cassava mosaic virus (ACMV, genus Begomovirus) have been studied by electron cryo- microscopy (cryo-EM) previously. While these investigations revealed some details about the geminate architecture, the interactions of capsid components have not yet been adequately modelled. The two incomplete icosahedral "heads" of the geminate particle are offset from one another and apparently make distinct CP:CP contacts at this region of the virion. Information regarding the nature of quasi- equivalent CP conformers or the sets of amino acid residues that mediate these interactions has not been forthcoming. Since the experimental results of these previous studies are not available in a public database, we were motivated to revisit the structure of MSV in order to obtain a 3D experimental density that might aid pseudo-atomic modelling. The MSV CP:ssDNA interaction has also been shown to be crucial for systemic movement through the host. Hence, quasi-atomic modelling may inform development of antiviral strategies which aim to interfere with virion assembly. MSV virions were isolated from the leaves of maize plants infected by agro-inoculation and visualized in both heavy metal stain and vitreous ice after they had been adsorbed to a thin-layer of continuous carbon to prevent virion aggregation. Virus preparations consisted of distinct CP assemblies consisting of multiples of the incomplete T=I icosahedral unit. Monopartite (icosahedral), bipartite (geminate), tripartite, and higher assemblies were observed suggesting the MSV CP is not only multifunctional but also structurally versatile being able to package ssDNA of variable sizes. Low-dose images were recorded on film, and 3D reconstruction of both monopartite and bipartite capsid species carried out using standard single-particle image processing methodology. The resolution of the bipartite reconstructions was 26 A for the negative-stain dataset, and 23 A for cryo-EM dataset, while the resolution of the monopartite reconstruction was estimated to be ~15 A. Comparative modelling of the MSV CP was undertaken using the pentamer (CPs) of Satellite tobacco necrosis virus (STNV) as a structural template. Correlation-based fitting of icosahedral and geminate atomic models that varied in geometric arrangement of MSV CPs allowed the geometric parameters of the bipartite capsid to be determined. Fitting ofMSV CPs into the EM densities informed our understanding of interfaces which allow the CP to self-associate, and showed that CPs is in fact displaced within the icosahedral geometry of the heads by a 10° rotation about the 5-fold axes of symmetry in comparison to STNV; hence, while quaternary structure of the pentameric capsomer is conserved between these viruses, the quaternary interactions between capsomers of the T=I unit has diverged considerably. This study shows that the offset between the geminate heads of the MSV virion is ~-11°, and that this geometry appears to arise owing to a distinct set of CP:CP interfaces which occur across the equator between two quasi-icosahedral heads and involve regions that would interact to form the CPs: CPs interfaces within each of the heads (across 2-fold and 3-fold symmetry axes). Notably this offset differs from that reported for ACMV, which has a reported offset of 20°. Additionally, the resolution afforded by the icosahedral monopartite reconstruction provided the first structural evidence to suggest that the calcium ion binding site of the STNV CPs (located on the CS axis) is likely to be conserved in MSV. This result suggests that in common with other plant viruses, depletion of calcium ions may be required for genome egress in a newly infected host cell. This study highlights the importance of future high-resolution studies of this unique virion morphology by both X-ray crystallography and cryo-EM.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/13389 |
| Date | January 2014 |
| Creators | Dent,Kyle Clayton |
| Contributors | Varsani, Arvind, Sewell, Trevor |
| Publisher | University of Cape Town, Faculty of Science, Department of Molecular and Cell Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
Page generated in 0.0025 seconds