The role of VPg in translation of calicivirus RNA
Daughenbaugh, Katie Finney
MetadataShow full item record
Molecular mechanisms of Norovirus replication remain for the most part undefined, primarily due to the lack of cell culture and small animal model systems. However, sequence comparisons and studies using cultivable caliciviruses have lead to the description of many features of the viral genome. Genomes are positive sense RNA, where the genome itself serves as mRNA for the production of viral protein. Additionally, viral RNA is covalently attached at the 5α end to the viral protein VPg. VPg is required for infectivity of the RNA by transfection, and removal of VPg by proteinase K treatment reduces the ability of the RNA to be translated in vitro. Because of these data, and because viral RNA is presumably not translated by an IRES mechanism, it has been suggested that VPg plays a role in translation of viral RNA. Studies described herein were initiated to investigate the potential role for Norwalk virus (NV) VPg in this process. It was found that NV VPg binds translation initiation factor 3 (eIF3) directly and in cell lysates, and is present in complexes with other eIFs including the cap-binding protein eIF4E, the large scaffolding protein eIF4G, the S6 ribosomal protein, and eIF2á, a component of the ternary complex. VPg also inhibits translation of reporter RNAs in vitro, suggesting that the interactions observed between VPg and eIFs are relevant to translation. Regions of VPg responsible for interactions with eIFs were mapped, and it was found that interaction between VPg and the 40S ribosome is most likely that which is responsible for translation inhibition of the reporter RNAs. VPg directly binds 40S ribosomal subunits by sucrose density gradient centrifugation, and this interaction is likely mediated by the central domain of VPg, similar to binding properties observed for the universally conserved factor eIF1A. Finally, a recently discovered, cultivable murine norovirus ₁ 1 (MNV-1) was used to ask if interactions between VPg and eIFs occur in infected cells. It was found that VPg of MNV ₁ 1 coprecipitates with eIF4GI, the d subunit of eIF3, and eIF4E from infected cells, and that this VPg has similar binding properties as the NV VPg. Together the data support the hypothesis that VPg plays a role in translation of viral RNA during infection, and suggets a third mechanism of ribosome recruitment dependent upon protein-protein interactions between VPg and eIFs. These studies also highlight the possibility of using MNV ₁ 1 as a molecular model for the study of human norovirus infection.