Project Title:

Characterisation of the mechanism of norovirus VPg-dependent RNA synthesis

Project Summary:

Noroviruses remain one of the most poorly characterised of all viruses yet, as the major cause of viral gastroenteritis, they have a huge economic impact.

Noroviruses are positive sense RNA viruses and the infectious viral RNA has a virus-encoded protein known as VPg attached to the 5’ end of the genome. VPg plays multiple roles in the viral life cycle, including translation, and most likely genome encapsidation. VPg is linked to viral RNA during genome synthesis in a process that is poorly understood but requires the addition of nucleotides to a highly conserved tyrosine residue in VPg. This process, known as VPg-guanylylation, is essential for norovirus replication.

Using reverse genetics and in vitro biochemical approaches, we have identified an RNA sequence/structure that functions as a template for the transfer of nucleotide to the viral protein VPg. This project will further dissect these interactions using both biochemical and molecular approaches. We will identify the regions of the viral genome that bind directly to viral replicase enzymes involved in VPg nucleotide transfer and also determine the secondary structure of this RNA using both SHAPE and our recently developed COMRADES method. Reverse genetics will be use to examine the role of the structure on viral replication and to identify mutations that attenuate norovirus replication. This work will use both murine and human noroviruses, building on recent developments in the field that allow for authentic human norovirus replication in cell culture using B cells and intestinal organoids.