This thesis presents structural studies on the plant virus Ryegrass mottle virus (RGMoV), the bacteriophage φCb5, and the icosahedral particles and octahedral crystal assembly of a bacteriophage MS2 coat protein mutant. In contrast to other sobemoviruses, the RGMoV coat protein is missing several residues in two of the loop regions important for capsid assembly. The first loop contributes to contacts between subunits around the quasi-three fold symmetry axis. The altered contact interface results in tilting of the subunits towards the quasi-threefold axis. The assembly of the T=3 capsid of sobemoviruses is controlled by the N-termini of the C subunits. The second and smaller RGMoV loop does not interact with the N-terminus of the C subunit as do the corresponding loops of other sobemoviruses. The loss of interaction has been compensated for by additional interactions between the N-terminal arms of RGMoV C subunits. The bacteriophage MS2 belongs to the Leviviridae family of small RNA phages. Covalent dimers of the coat protein with insertions in the surface loops are known to be highly immunogenic epitope carriers. We crystallized the icosahedral particle assembled from covalent coat protein dimers in space group P213. At 4.7Å resolution the structure resembles the wildtype MS2 virion except for the intersubunit linker regions. The covalent dimer also crystallized in the cubic space group F432. The organization of the asymmetric unit in combination with the F432 symmetry results in an arrangement of subunits that corresponds to T=3 octahedral particles. Our crystal structure of the bacteriophage φCb5 capsid showed that it is stabilized by four calcium ions per icosahedral asymmetric unit. One ion is located between the quasi-threefold related subunits and is important for formation of a network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilized the contacts within the coat protein dimer. There was electron density of three putative RNA nucleotides per icosahedral asymmetric unit in the φCb5 structure. The nucleotides mediated contacts between two subunits forming a dimer and a third subunit in another dimer. On the basis of these findings, we have proposed a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The structure of RGMoV increases our understanding of mechanisms controlling sobemovirus assembly. This knowledge could be used to create genetically modified plants resistant to sobemovirus infection. The modified capsids of leviviruses can be used in immunization and as vehicles for gene or therapeutic compound delivery.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-99933 |
| Date | January 2009 |
| Creators | Plevka, Pavel |
| Publisher | Uppsala universitet, Strukturell molekylärbiologi, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 627 |
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