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  • 1
    Online Resource
    Online Resource
    Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening
    American Society for Microbiology ; 2019
    In:  Journal of Virology Vol. 93, No. 1 ( 2019-01)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 93, No. 1 ( 2019-01)
    Abstract: Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together, these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules. IMPORTANCE Norovirus is one of the leading causes of food-borne illness worldwide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed, we performed genome-wide CRISPR activation screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes that could block murine norovirus replication in human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data are a resource for those studying noroviruses, and we provide a robust approach to identify novel antiviral genes.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.01324-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 1495529-5
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  • 2
    Online Resource
    Online Resource
    Selective Polyprotein Processing Determines Norovirus Sensitivity to Trim7
    American Society for Microbiology ; 2022
    In:  Journal of Virology Vol. 96, No. 17 ( 2022-09-14)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 96, No. 17 ( 2022-09-14)
    Abstract: Noroviruses are a leading cause of gastroenteritis worldwide, yet the molecular mechanisms of how host antiviral factors restrict norovirus infection are poorly understood. Here, we present a CRISPR activation screen that identifies mouse genes which inhibit murine norovirus (MNV) replication. Detailed analysis of the major hit Trim7 demonstrates a potent inhibition of the early stages of MNV replication. Leveraging in vitro evolution, we identified MNV mutants that escape Trim7 restriction by altering the cleavage of the viral NS6-7 polyprotein precursor. NS6, but not the NS6-7 precursor, directly binds the substrate-binding domain of Trim7. Surprisingly, the selective polyprotein processing that enables Trim7 evasion inflicts a significant evolutionary burden, as viruses with decreased NS6-7 cleavage are strongly attenuated in viral replication and pathogenesis. Our data provide an unappreciated mechanism of viral evasion of cellular antiviral factors through selective polyprotein processing and highlight the evolutionary tradeoffs in acquiring resistance to host restriction factors. IMPORTANCE To maximize a limited genetic capacity, viruses encode polyproteins that can be subsequently separated into individual components by viral proteases. While classically viewed as a means of economy, recent findings have indicated that polyprotein processing can spatially and temporally coordinate the distinct phases of the viral life cycle. Here, we present a function for alternative polyprotein processing centered on immune defense. We discovered that selective polyprotein processing of the murine norovirus polyprotein shields MNV from restriction by the host antiviral protein Trim7. Trim7 can bind the viral protein NS6 but not the viral precursor protein NS6-7. Our findings provide insight into the evolutionary pressures that define patterns of viral polyprotein processing and uncover a trade-off between viral replication and immune evasion.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/jvi.00707-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 1495529-5
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  • 3
    Online Resource
    Online Resource
    CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
    American Society for Microbiology ; 2018
    In:  mBio Vol. 9, No. 3 ( 2018-07-05)
    Details
    In: mBio, American Society for Microbiology, Vol. 9, No. 3 ( 2018-07-05)
    Abstract: Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen’s key virulence factors.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01003-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 2557172-2
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  • 4
    Online Resource
    Online Resource
    High-Throughput CRISPR Screens To Dissect Macrophage- Shigella Interactions
    American Society for Microbiology ; 2021
    In:  mBio Vol. 12, No. 6 ( 2021-12-21)
    Details
    In: mBio, American Society for Microbiology, Vol. 12, No. 6 ( 2021-12-21)
    Abstract: Shigellosis causes most diarrheal deaths worldwide, particularly affecting children. Shigella invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To understand how Shigella rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri -infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced proinflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small-molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the in vivo physiological state of macrophages postinfection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies. IMPORTANCE Treatment for shigellosis is becoming increasingly difficult as resistance to antibiotics becomes more prevalent. One way to prevent this significant public health problem from developing into a full-blown crisis is to approach shigellosis intervention from the point of view of the host. So far, little is known about the specific biological pathways that might be modulated in macrophages, sentinel cells of the innate immune system, to strengthen the response to Shigella infection. In this work, we conducted CRISPR screens to comprehensively decipher the complexity of macrophage- Shigella interactions and to discover new potential therapeutic interventions against Shigella flexneri infection. Our work highlights systematic genetic perturbation strategies to provide direct causal evidence showing how intracellular pathogens manipulate innate immune cells.
    Type of Medium: Online Resource
    ISSN: 2150-7511
    URL: Article
    DOI: 10.1128/mBio.02158-21
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 2557172-2
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