Project description:Norovirus, a member of the Caliciviridae family, is a positive-sense single-stranded RNA (+ssRNA) virus and a leading cause of acute gastroenteritis worldwide, especially affecting young children and the elderly. Due to a lack of specific treatment, norovirus infection causes 200,000 deaths each year and imposes an economic burden exceeding $60 billion. To address this challenge and develop medicines and diagnostics, further research into norovirus reproduction is needed. Norovirus uses a nucleotidylated protein, known as VPg (viral protein genome-linked), to initiate genome replication, by acting as a replication primer. The genomic RNA of norovirus is linked by a phosphodiester bond between the 5’-terminal guanosine (norovirus) and the phenolic oxygen of a tyrosine residue (Y27 for norovirus). In this work we demonstrate the synthesis of peptides derived from guanylylated norovirus VPg and their use in proteomic studies of norovirus replication.
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.
Project description:Gene expression analysis of GI human norovirus (HuNoV) replicon in human gastric tumor cells (HGT-NV), IFN-cured population of replicon-harboring HGT-1 cells (HGT-Cured) and wild-type parental HGT-1 cells (HGT) to provide insight into the cellular factors promoting human norovirus replication in vitro.
