Characterisation of the Mechanism of Norovirus VPg-Nucleotidylylation

Noroviruses, which belong to the *Caliciviridae* family, have a substantial yearly impact on the global economy of about $60 billion and cause about 200,000 deaths per annum in children under the age of 5. Despite causing most cases of viral gastroenteritis, noroviruses remain poorly characterised compared to other viruses. Infectious norovirus RNA is covalently linked to VPg (viral protein genome-linked) at the 5’ end of the genome through VPg-mediated RNA priming of viral RNA synthesis. While poorly understood mechanistically, this process is reliant on the addition of a nucleotide to a highly conserved tyrosine residue within VPg - this process is referred to as VPg nucleotidylylation and is catalysed by the viral polymerase (NS7). Using murine norovirus (MNV) as the primary model, this project sought to better understand this key stage of the norovirus life cycle, providing insight into a previously ill-characterised crucial stage of the viral life cycle and may help to identify key points which could be therapeutically targeted. Mass spectrometric analysis of the 5’ end of infectious norovirus RNA purified from infected cells, confirmed that VPg is genome-linked via a GDP moiety at a highly conserved tyrosine residue. These results however are contradicted by the state of the art on calicivirus VPg nucleotidylylation and has, to date, not been replicated *in vitro*. Therefore, developing a specific assay to measure *in vitro* VPg nucleotidylylation producing physiologically relevant readouts laid at the core of this project. Subsequently, this, for the first time, brought previous *in vivo*, genomic and *in vitro* observations of VPg nucleotidylylation in agreement and enabled me to study this reaction. I identified negative sense subgenomic RNA (-sgRNA), magnesium ions (Mg²⁺) and the viral protease (NS6) as essential components that facilitate specific and physiologically relevant VPg nucleotidylylation. Further, I showed precursor forms of NS6, NS56 and NS67, to stimulate the reaction more efficiently than mature NS6. Mutations within potential RNA binding regions of NS6 affected VPg nucleotidylylation *in vitro*, suggesting a potential role in the reaction for it as stabilizing the nucleotidylylation complex, through binding the RNA template. I identified a region within the -sgRNA of MNV, spanning region in the 3’ end of open reading frame 3 (ORF3) and the untranslated region (UTR), to be both sufficient and necessary for stimulating VPg nucleotidylylation. Through subsequent serial truncations, internal deletions and point mutations, I was then able to refine this understanding. My findings indicate VPg nucleotidylylation to be reliant on three distinct structural elements within this region, one of them a small, predicted stem-loop in the UTR. Utilising a panel of MNV mutants within the 3’ region of the genome, previously found to affect virus viability, I observed a clear correlation between virus viability and the negative sense 3’ region’s ability to enhance VPg nucleotidylylation. Lastly, I demonstrated the ORF3 UTR region of HuNoV RNA to be able to template MNV VPg nucleotidylylation, suggesting template and positional conservation between MNV and HuNoV. In summary, this work provides new key insights into VPg nucleotidylylation - a previously poorly characterised, but vital, process of norovirus replication and may pave the way for further exploration of this process for therapeutic targeting.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Pinckert, Malte

Advisor dc:contributor.advisor

• Goodfellow, Ian

• Caliciviridae
• guanylylation
• HuNoV
• MNV
• murine norovirus
• Norovirus
• nucleotidylylation
• viral life cycle
• viral replication
• VPg

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0970b5d1-5655-42d3-845d-d7848bb9d67d/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107405

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366534