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Adenovirus-cell interactions for recognition signalling and entry probed by FRET FLIM and time-resolved anistropy

Reference	BB/C515039/1
Principal Investigator / Supervisor	Professor George Santis
Co-Investigators / Co-Supervisors	Professor Marisa Martin-Fernandez, Professor Madeline Parsons, Professor Brian Sutton
Institution	King's College London
Department	Grants Payments
Funding type	Research
Value (£)	226,961
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/08/2008
Duration	36 months

Abstract

Virus infection requires entry of virus particles into target cells, transport to the nucleus and viral genome replication. Virus interactions with cell receptors is a central step in productive infection as it determines not only virus cell attachment and internalisation but also initiates subsequent steps in virus life cycle. These events require the generation of appropriate signals to be generated by the interaction between virus and its receptors. Adenoviruses are small non-enveloped viruses that cause a wide range of human infections and their tropism is predominantly determined by the virus cell receptor interaction. Subgroup B and C adenoviruses attach to the cell surface by binding to coxsackievirus-adenovirus receptor (CAR). Following attachment the virus interacts with alpha v beta 3 5 integrin molecules and this promotes virus endocytosis and initiates cell signalling that is necessary for intracellular virus trafficking. Sub-group B viruses that do not bind to CAR have been recently shown to utilise CD46 as their cell receptor. Despite these advances, major questions relating to the biology of adenovirus-cell receptor interactions remain unresolved. This proposal aims at addressing two central questions: 1) How adenoviruses binding to its attachment and internalisation receptors generates the necessary signals for efficient virus uptake, intracellular trafficking and capsid disassembly. 2) How events that follow adenovirus-receptor interaction differ from those initiated by the interaction between the same receptors and their natural ligands. In order to address these questions we have developed novel strategies that involve the use of Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging Microscopy (FLIM) and time resolved anisotropy. For these experiments we have exploited the unique power of the SR microfluorimeter and confocal microscope at Daresbury Laboratory. These assays, which were developed in collaboration with Dr Marisa Martin-Fernandez at Daresbury, will enable us to study in detail intracellular trafficking of different Ad and the disassembly of their capsid during uptake in living cells. This powerful technique offers advantages over current methodologies in that it gives multiple assessments in real time on living cells regarding the state of the adenoviral structure, the pH microenvironment and the kinetic rate at which the dissociation events occur without the need for invasive or destructive biochemical analysis. Understanding the way in which adenovirus behaves at the cell surface and the role of intracellular signalling in adenovirus internalisation is critical to understanding mechanisms of viral infectivity. Unravelling the differences in the interaction between receptors and their natural vs pathological ligands will enhance our understanding of protein-protein interactions and the signals that they generate. A greater understanding of virus-receptor interactions will allow the development of more efficient vector systems for gene delivery and therapeutic use.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Pharmaceuticals
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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