Characterisation of the novel mechanism of protein-directed translation initiation used by caliciviruses

Abstract

Human noroviruses of the Caliciviridae family are the major cause of acute gastroenteritis in the developed world, but little is known about their molecular mechanisms of translation and replication. Since human noroviruses are not cultivatable in vitro, most molecular studies have therefore been based on the cultivatable murine norovirus (MNV) or feline calicivirus from the Vesivirus genera. Calicivirus translation is dependent on the viral VPg protein covalently linked to the 5’ end of viral RNA and removal of the VPg from calicivirus RNA results in loss of infectivity. VPg functions as a ‘cap substitute’ by interacting with eIF4E to recruit translation initiation factors (eIFs). Given that VPg plays a critical role in the virus life cycle, it is potentially a good anti-viral target. As an initial attempt to identify ways of targeting VPg as an anti-norovirus therapy, we used phage display to identify peptides with an affinity for VPg. Despite repeated attempts however, we failed to generate a peptide that had clear specificity for the calicivirus VPg. Since VPg has ability to bind the cap binding protein eIF4E, m7-GTP Sepharose affinity chromatography was used to isolate eIF4E and the associated VPg. Similar to the analysis of the MNV VPg-translation initiation factor complex, this method successfully isolated not only the human norovirus VPg, but also isolated the entire eIF4F complex and PABP from a cell based-norovirus replicon system. Human norovirus and MNV VPg proteins share 54% amino acid identity and MNV has been proposed as a good model for human norovirus. To begin structural and functional analysis of the human norovirus VPg in the context of an authentic viral life cycle, we introduced the human norovirus VPg into an infectious clone of MNV, to generate a chimera containing all the MNV non-structural and structural proteins but with the human norovirus VPg. Whilst the insertion of the human norovirus VPg into the MNV genome did not affect polyprotein processing, we were unable to recover infectious viral particles. Since the requirement for other cellular factors in the VPg-dependent translation is not fully understood, we used “tandem affinity purification” to further define the components of the initiation factor complex associated with VPg. Our data demonstrated that VPg associates with both canonical initiation factors and possibly non-canonical initiation factors. Further investigation suggested that the scaffold protein eIF4G, which bridges the interaction between PABP and eIF4E, binds directly to VPg via its central domain. To determine the function of eIF4G in the norovirus life cycle, we have silenced eIF4G isoforms in 293T cells using specific siRNAs and transfected them with infectious VPg-linked viral RNA. The siRNA-mediated knockdown of either eIF4G isoform resulted in a 9 fold reduction in virus titre, as well as reduction of viral RNA and protein synthesis, confirming that eIF4G has a functional role in norovirus life cycle. Furthermore, it is not known if the eIF4G-eIF4E interaction is not limiting for MNV translation. Thus, we altered the availability of eIF4E to bind eIF4G by overexpressing wild type or non-phosphorylatable mutant forms of the eIF4E binding protein 1 (4E-BP1) in cells. Overexpression of either the wild type 4E-BP1 or the non-phosphorylatable 4E-BP1 mutant in MNV infected cells showed no significant change in viral RNA, virus titre or protein expression suggesting that the eIF4G-eIF4E interaction in a VPg-translation initiation complex is not limiting for MNV translation. In addition to studying the interaction between VPg and the eIFs, we also examined the effect of mutations in VPg on virus viability and ability to bind the eIF4F complex. A number of mutants were generated in MNV infectious clone to identify residues that play a role in calicivirus translation. The mutational analysis of MNV VPg indicated the C-terminus of VPg is essential for the association of VPg with the components of eIF4F complex and virus viability. This work provides new insights on the interaction between noroviruses and the host cell, as well as the novel mechanisms that viruses use to synthesis their proteins.