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:Murine norovirus is genetically similar to human norovirus, and offers both an efficient in vitro cell culture system and animal model by which to investigate the molecular basis of replication. Here, we present a detailed global view of host alterations to cellular pathways that occur during the progression of a norovirus infection. This was accomplished for both RAW264.7 (RAW) cells, an immortalized cell line widely used in in vitro replication studies, and primary bone marrow-derived macrophages (BMDM), representing a permissive in vivo target cell in the host. Murine norovirus replicated in both cell types, although detected genome copies were approximately one log lower in BMDM compared to RAW cells. RAW and BMDM cells shared an IRF3/7-based IFN response that occurred early in infection. In RAW264.7 cells, transcriptional upregulation and INF-ß expression were not coupled, in that a significant delay in the detection of secreted INF-ß was observed. In contrast, primary BMDM showed an early upregulation of transcripts and immediate release of INF-ß that might account for lower virus yield. Differences in the transcriptional pathway responses included a marked decrease in expression of key genes in the cell cycle and lipid synthesis pathways in RAW264.7 cells compared to that of BMDM. Our comparative analysis indicates the existence of varying host responses to virus infection in populations of permissive cells. Awareness of these differences at the gene level will be important in the application of a given permissive cell culture system to the study of norovirus immunity, pathogenesis, and drug development.
Project description:The goal of this study was to evaluate the transcriptional response of 4 human duodenal enteroid lines on monolayers to norovirus infections (GII.4). Enteroids were plated as monolayers in Intesticult (Stem Cell Technologies) proliferation medium. After 1 day of cell growth as a monolayer, the proliferation medium was changed with differentiation medium for 5 days. Five-day-differentiated monolayers were washed and were either mock-infected or inoculated with human norovirus supplemented with 500 μM glycochenodeoxycholic acid (GCDCA), for 1 to 2 h at 37°C. Total RNA was extracted using the Qiagen RNeasy kit and paired-end Illumina sequencing was performed.
Project description:Norovirus replication is accomplished by the use of a number of viral proteins derived from a long polyprotein. These components are named from NS1/2 to NS7. Having previously determined that the incorporation of FLAG epitope tags into certain positions in the norovirus genome was tolerated (Thorne et. al. 2012 J. Virol)