Abstract
Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide. Despite advances in HuNV cell culture the virus remains difficult to study in the laboratory. Murine norovirus (MNV) is routinely utilised as a model virus for the study of HuNV replication and pathogenesis. MNV has been shown to manipulate the host cell to induce a G0/G1 cell cycle arrest and gain a beneficial replication environment. Expression of the viral protein genome-linked (VPg) protein of MNV is sufficient to induce cell cycle arrest. Manipulation of the cell cycle is independent of the known translation and replication functions of VPg and can be induced with the first 62 N-terminal amino acids. This research aimed to further characterise the interaction between norovirus VPg proteins and the cell cycle.
All viruses in the Caliciviridae family encode a VPg protein however it is not known if the G0/G1 arrest induced by MNV VPg is conserved in other calicivirus VPg proteins. The cell cycle effect of representative VPg proteins from five norovirus genogroups and four other Caliciviridae genera were analysed. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins and representative VPg proteins from the Lagovirus and Sapovirus genera. These findings show that manipulation of the host cell cycle is a highly conserved function of the VPg proteins of caliciviruses and consequently is likely to be important for the viral lifecycle.
Alignment of VPg protein sequences revealed a conserved KGKxKxGRG N-terminal motif with high levels of positively charged amino acids. To test the importance of this region for inducing a G0/G1 arrest, truncated or mutated MNV VPg constructs were tested. Removal of the N-terminal motif prevented an accumulation of cells in the G0/G1 phase confirming the importance of this region. Single point or triple mutations of lysine and arginine significantly reduced the accumulation of cells in the G0/G1 phase but did not completely inhibit the arrest confirming the role of positively charged amino acids in this activity. However, expression of the N-terminal motif fused to EGFP was not able to manipulate the cell cycle, indicating that there are additional elements of MNV VPg that contribute to an arrest.
Bioinformatic and literature analysis of the N-terminal motif of MNV VPg indicated three activities that could have a role in inducing a G0/G1 phase arrest. The N-terminal region was predicted to have RNA binding activity, function as a nuclear localisation sequence and interact with eIF4E. Gel shift analysis showed that MNV VPg and HuNV VPg bind to RNA in a non-specific manner and the lysine and arginine residues within the N-terminal motif were shown to be important for this activity. No evidence was found for nuclear localisation of MNV VPg. Infection of cells with MNV resulted in a dysregulation of eIF4E leading to an increase in nuclear eIF4E levels. Changes in the nuclear/cytoplasmic ratios of eIF4E is a possible mechanism by which VPg could induce a G0/G1 phase arrest.
This thesis provides evidence that manipulation of the host cell cycle by VPg proteins is a common feature of caliciviruses that involves a positively charged motif at the N-terminus of VPg. Both RNA binding and dysregulation of eIF4E were identified and remain as possible mechanisms by which MNV VPg may manipulate the cell cycle.