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Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

ABSTRACT: Transcription of interferons upon viral infection is critical for cell-intrinsic innate immunity. This process is influenced by many host and viral factors. To identify host factors that modulate interferon induction within cells infected by influenza A virus, we developed CRISPR with Transcriptional Readout (CRITR-seq). CRITR-seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor complex increases both flu transcription and interferon expression. We find that the process of flu transcription, both in the presence and absence of viral replication, is a key contributor to interferon induction. Taken together, our findings highlight innate immune ligand concentration as a limiting factor in triggering an interferon response, identify NELF as an important interface with the flu life cycle, and validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE281730 | GEO | 2024/11/14

REPOSITORIES: GEO

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Project description:Peste des petits ruminants virus (PPRV) infection causes highly contagious and severe disease in domestic and wild ruminants. It is widely reported that PRRV infection causes considerable innate immunosuppression in its host and promotes viral replication. However, how does the host rescue the innate immune response to counteract this immunosuppression during viral replication is poorly understood. The goat fetal fibroblasts (GFFs) have been commonly used as host-original cells to investigate the pathogenesis of PPRV. To explore the mechanisms of how host counteracts PPRV-mediated innate immunosuppression, a high-throughput quantitation proteomic approach (iTRAQ in conjunction with LC-MS/MS) was used to investigate the proteome landscape of GFFs in response to PPRV infection. Eventually, the proteomic analysis gained 497 up-regulated proteins and 358 down-regulated proteins (PPRV-infected cells versus mock-infected cells). The complement and coagulation cascades, protein digestion and absorption, and cytokine-cytokine receptor interaction pathways were significantly regulated in response to PPRV infection. ErmineJ analysis of the differentially expressed proteins (DEPs) identified the significantly enriched GO categories for response to interferon-gamma and positive regulation of ERK1 and ERK2 cascade. In addition, many immune related proteins, such as interferon gamma, 2'-5'-oligoadenylate synthase-like protein, toll-like receptor 9, toll-like receptor 6, NOD1, plasminogen activator inhibitor 1, and Wnt-5a were significantly upregulated. This suggested that the innate immune response was triggered during PPRV infection in GFFs. We subsequently identified that the E3 ubiquitin ligase FANCL was critically involved in regulation of the innate immune response during PPRV infection. FANCL inhibited PPRV infection by enhancing type I interferon (IFN) and interferon-stimulated genes (ISGs) expression. Further study indicated that FANCL induced type I IFN production by promoting TBK1 phosphorylation, and therefore impairing PPRV-mediated immunosuppression and revealing an antiviral function against PPRV.

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Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

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