Abstract
Caliciviruses cause important diseases of humans and animals; noroviruses are responsible for outbreaks of gastroenteritis in man, and feline calicivirus (FCV) causes an upper respiratory tract infection in cats. Since 2003, murine norovirus (MNV) has been used as a model for norovirus infection since human noroviruses are unable to replicate in tissue culture. Caliciviruses have a positive-sense RNA genome of about 7 to 8 kb, with a viral protein, VPg, covalently linked to the 5' end of the RNA. The VPg acts as a novel proteinaceous ‘cap substitute’ and interacts with the cap-binding protein, eIF4E. To further analyse the requirement of translation initiation factors for calicivirus translation, I have investigated the effect of FCV and MNV infection on eIF4E and its regulator, 4E-BP1. I found that FCV and MNV infection have different effects on the phosphorylation status of eIF4E and its regulator, 4E-BP1. For both viruses, eIF4E is phosphorylated and 4E-BP1 is dephosphorylated during the course of infection. This would be expected to inhibit host cell protein synthesis. This has led me to investigate the signalling pathways involved in mediating these effects. Using specific inhibitors I have shown that phosphorylation of eIF4E during FCV and MNV infection occurs through activation of the MEK/MAPK pathway, while 4E-BP1 phosphorylation occurs through PI3K/mTOR activation. When I use specific inhibitors are used to block either the p38 or MEK pathways, replication of MNV is inhibited. However, FCV replication is only inhibited by MEK inhibition. In addition, eIF4E phosphorylation is prevented by blocking p38 but not MEK. This suggests that the changes in eIF4E phosphorylation may not affect the interaction between eIF4E and VPg or be important for FCV and MNV replication. These changes may therefore only be involved in host-cell shut-off. In contrast, inhibition of the mTOR pathway significantly inhibited both FCV and MNV replication, suggesting that the phosphorylation status of 4E-BP1 during infection is important for virus replication. I hypothesise that the change in phosphorylation status of 4E-BP1 may lead to the inhibition of eIF4F complex formation and therefore calicivirus translation; this may affect the switch from translation to replication of the viral RNA. I propose that the mTOR pathway could be a target for antiviral therapy. I have also further investigated the exact requirements for components of the eIF4F cap-binding complex for translation initiation on FCV and MNV mRNA. I have shown that a small-molecule inhibitor of the eIF4E—eIF4G interaction has a greater inhibitory effect on FCV than MNV replication, demonstrating a requirement by FCV for eIF4E and an intact eIF4G for viral RNA translation.