Project description:The norovirus VPg protein is covalently linked to the viral genome in place of a 5' cap, and functions as a cap-substitute, capable of interacting with translation initiation factors. Following on from our previous study (Chung et. al. 2014, J. BIol. Chem.) we wished to determine the interactome of human norovirus VPg, and compare that of murine norovirus VPg. We had previously demonstrated that mutation of the penultimate C-terminal phenylalanine residue in murine norovirus VPg greatly reduced initiation factor binding (F123A). Insertion of the equivalent mutation into human norovirus (F137A) also reduced initiation factor binding. Affinity purification of wild-type of mutant human and murine norovirus VPg was accomplished using GFP-tagged VPg transfected into SILAC-labelled human HEK-293T cells.
