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Title: Identification of RNA-protein interactions involved in the norovirus life cycle
Author: Urena Gonzalez, Luis
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Members of the Caliciviridae family, including murine norovirus (MNV), contain conserved RNA secondary structure elements located at the 5' and 3' regions of the viral genome. The 5' extremity of the genomic (5'G) and subgenomic (5'SG) RNAs, as well as the 3' extremity (3'Ex), are believed to be involved in many aspects of calicivirus life cycle. This project was designed to identify proteins interacting with the extremities of MNV genome, as this would include proteins involved in viral translation and replication. We used a riboproteomics method to isolate RNA-binding proteins by RNA affinity chromatography and mass spectrometry from RAW264.7 S-100 extracts and rabbit reticulocyte lysates (RRL). Combining data sets obtained from RRL and S-100 lysates; various unique cellular host factors were identified using the 5'G, 5'SG, and 3'Ex RNA targets. These included several RNA-binding proteins involved in other positive-strand virus replication but not reported for Caliciviridae family, including Y-box binding protein 1 (YBX-1), ATP- dependent RNA helicase (DDX3), and Heat Shock Protein 90 (HSP90). Other identified proteins isolated as binding to the MNV genome included known calicivirus interacting proteins such as La protein (La), poly(rC) binding protein 1 (PCBP) and polypyrimidine tract binding protein (PTB). The colocalisation of some of the proteins was confirmed by confocal microscopy. The functional role of a subset of identified proteins was further analysed by using RNAi and specific protein inhibitors. These results indicate that knockdown of La, PTB, YBX-1, DDX3 and HSP90 dramatically reduces viral titre and RNA replication. Confocal microscopy showed that some of these host factors redistribute to the site of infection colocalising with MNV replication components. The use of DDX3 and HSP90 inhibitors demonstrated a significant effect on infectious virus production confirming that these factors play a crucial role in the virus life cycle. This work highlights how screening of RNA-binding proteins in the context of MNV by a riboproteomics approach could well provide insights in the mechanisms for the regulation of viral translation and replication, and could potentially pinpoint new drug targets for these important pathogens.
Supervisor: Goodfellow, Ian Sponsor: Secretaria Nacional de Ciencia ; Tecnologia e Innovacion ; Panama
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available