Architecture and assembly of maize streak virus: insights from 3D electron microscopy

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dc.contributor.advisorVarsani, Arvinden_ZA
dc.contributor.advisorSewell, Trevoren_ZA
dc.contributor.authorDent,Kyle Claytonen_ZA
dc.date.accessioned2015-07-08T06:22:39Z
dc.date.available2015-07-08T06:22:39Z
dc.date.issued2014en_ZA
dc.descriptionIncludes bibliographical references.en_ZA
dc.description.abstractMaize 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.en_ZA
dc.identifier.apacitation (2014). <i>Architecture and assembly of maize streak virus: insights from 3D electron microscopy</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/13389en_ZA
dc.identifier.chicagocitation. <i>"Architecture and assembly of maize streak virus: insights from 3D electron microscopy."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2014. http://hdl.handle.net/11427/13389en_ZA
dc.identifier.citation 2014. Architecture and assembly of maize streak virus: insights from 3D electron microscopy. Thesis. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. http://hdl.handle.net/11427/13389en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Dent,Kyle Clayton AB - 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. DA - 2014 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2014 T1 - Architecture and assembly of maize streak virus: insights from 3D electron microscopy TI - Architecture and assembly of maize streak virus: insights from 3D electron microscopy UR - http://hdl.handle.net/11427/13389 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/13389
dc.identifier.vancouvercitation. Architecture and assembly of maize streak virus: insights from 3D electron microscopy. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2014 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/13389en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherStructural Biologyen_ZA
dc.titleArchitecture and assembly of maize streak virus: insights from 3D electron microscopyen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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