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 (MNV) is genetically similar to human norovirus (HuNoV), 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 the cellular alterations that occur during the progression of a norovirus infection. The transcriptome of a synchronously infected population of MNV-infected murine macrophage-like cell line (RAW264.7) was determined at 8, 14, and 20 hours post infection. The cellular genetic response was analyzed both globally and by specific pathways. Viral replication was monitored by RNA-seq and quantitative real-time PCR in context of the cellular phenotypic response. The majority of transcriptionally up regulated genes were related to the IFN response. Additionally, there was a global increase in gene transcripts associated with immune response and inflammation. A transcriptional decrease was observed across many cellular processes, but particularly for genes involved in lipid homeostasis and cell cycle. The peak of the transcriptional immune response correlated with detected viral genome copies and changes in cellular phenotype including nuclear condensation. A more complete understanding of host response to norovirus infection will help to highlight the cellular pathways critical for a more effective immune response as well as those that may be exploited by the virus for therapeutic development.
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)
Project description:Noroviruses have been widely recognized for their importance as causative agents of non-bacterial gastroenteritis. Mouse norovirus is the only representative of the norovirus genus, family Caliciviridae, able to grow in cell culture. The aim of this study is to describe the differences in the expression profiles of MNV-1 and mock-infected macrophages (RAW 264.7 cells), in order to better understand the response of the host cell to norovirus infection.
Project description:Noroviruses have been widely recognized for their importance as causative agents of non-bacterial gastroenteritis. Mouse norovirus is the only representative of the norovirus genus, family Caliciviridae, able to grow in cell culture. The aim of this study is to describe the differences in the expression profiles of MNV-1 and mock-infected macrophages (RAW 264.7 cells), in order to better understand the response of the host cell to norovirus infection. Experiment Overall Design: This study compares two type of samples (MNV-infected and mock-infected RAW cells) in biological triplicates respectively. The MNV stock used for infection was obtained in the same cell line, and purified with a sucrose cushion in order to account for expression changes caused by virus infection only.
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.