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Contenuto archiviato il 2024-04-30

Structures of double-stranded RNA viruses

Obiettivo



OBJECTIVES
Summary: The objective is to choose a complex biological system that is already well studied in Europe and to develop a collaboration that will bring together a range of scientific disciplines including two of the main techniques of structural biology (cryo electron microscopy and X-ray crystallography).
The biological system we have chosen is double stranded RNA viruses. This diverse range of viruses infects a wide range of organisms, and although we suspect that there are certain fundamental similarities in key aspects of their life-cycle, it is very difficult at present to assess this. We have therefore selected two complimentary virus systems to study, a genera of mammalian viruses orbiviruses (family Reoviridae) and, at the opposite edge of the evolutionary spectrum, bacteriophage 06. We will investigate the ability of these viruses to package, with complete reliability, one copy of each of a number of genome segments into each particle. The structural basis of this astonishing feat of molecular recognition is unknown. These virus capsids are at the limit of complexity of macro-molecular systems that can be analysed by present crystallographic methods and present fascinating opportunities for developing a biological understanding since they are far from the inert containers that such capsids are often seen as. They are dynamic, in the conformational changes that accompany the capsid maturation. Also, most interestingly, the capsid (or a core particle derived from the capsid) comprises a transcriptional machine. Although it functions normally in the cytoplasm of the infected cell we can activate the machine in-vitro and even in the crystal. The symmetry of the transcriptional complex is lower that icosahedral so the proposal will combine studies of icosahedrally averaged structures, studies of isolated components and studies also using novel methods of reconstructing non-symmetric systems.
The work will therefore focus on developing a biological understanding of complex molecular assemblies. This biological aim will drive the development of structural methodologies and the techniques for linking these methods together to maximize the potential for synergy.
Methodological developments anticipated:
X-ray crystallography:
- push data collection at European resources to the limit, keeping an international edge
- develop methods for reliably describing poorly ordered structures by X-ray crystallography
- improve methods of comparing information from EM and crystallography - develop methods for refining structures violating crystallographic symmetry
Cryo electron microscopy.
- develop methods for optimal reconstructions from non-symmetric structures - optimize analysis of systems with mixed symmetry
- continue development of time-resolved methods in EM to analysis the activation of transcription
Biological prospects: The dsRNA viruses include a number of important pathogens and are the subject of active study worldwide. Despite strong competition from America the crystallographic analyses we have made on an orbivirus place us ahead of groups elsewhere, providing us with the largest macro-molecular assembly yet visualized at atomic detail. This has also allowed us to locate the polymerase complexes and substantial portions of genomic RNA within the capsid. This will provide us with a firm framework, to which results from other techniques and viruses can be related. This application is designed to build a supra-national collaboration based on the particular strengths of the partners in virology, electron microscopy and crystallography. We will invoke a variety of advanced structural methods with the aim of producing a uniquely detailed and yet comprehensive understanding of several key stages in the viral life-cycle; characterizing a macro-molecular machine that could ultimately be put at the service of a variety of biotechnologies.

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Coordinatore

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Contributo UE
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Indirizzo
South Parks Road
OX1 3QT OXFORD
Regno Unito

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