Studies on the mechanism of norovirus RNA synthesis

Abstract

Noroviruses are common pathogens associated with gastroenteritis in humans. As single-stranded positive-sense RNA viruses, noroviruses achieve their genome replication through the synthesis of negative strand RNA intermediates that template the generation of new positive strand RNA genomes. The murine norovirus (MNV) RNA-dependent RNA polymerase (NS7) protein which is a key player in catalysing this process, has been characterised in this study. Tandem affinity purification of MNV NS7 was performed in order to identify host cell factors which interact with the NS7 protein. Proteomic analysis demonstrated that guanosine monophosphate reductase and N(2)- dimethylguanosine tRNA methyltransferase could potentially interact with the MNV NS7. Furthermore, affinity selection of small peptides which specifically bind to the NS7 was carried out by using the phage display technique in an effort to generate peptide inhibitors. Peptide phage with two different conserved motifs and several peptide phage pools with binding activity to the NS7 were successfully identified. However, further cross-binding analysis using ELISA demonstrated that these peptide phage possibly bound non-specifically to the MNV NS7. An optimised RNA based reverse genetics system and reporter-tagged replicon system for MNV were also successfully developed and used to quantify the effects of specific mutations in the MNV genome on viral replication in tissue culture. Using these newly developed systems, the functional role of a small stem loop structure located upstream of the start site of the subgenomic RNA was characterised. This was identified as the potential viral RNA polymerase promoter responsible for subgenomic RNA synthesis. Furthermore, identification and characterisation of this stem loop using mutations suggested the potential involvement of long range RNA-RNA interactions (on negative strand RNA) in regulating the norovirus subgenomic RNA synthesis. Overall, this study has unveiled the importance of protein-protein interactions and RNA-protein interactions in regulating norovirus replication. These interactions could provide interesting targets for antiviral therapeutic intervention in the future.