Functional characterisation of a Viperin knockout fish cell line
Résumé
The antiviral response is mediated through the expression of a wide range of interferon-stimulated genes (ISGs). The radical S-adenosyl-methionine domain-containing protein 2 (Rsad2), also known as Viperin, is a well-known ISG-encoded protein with a potent antiviral activity across species and cell types. In mammals, Viperin exerts its function through different pathways: (1) it catalyses the conversion of cytidine triphosphate (CTP) to its analogue ddhCTP, which inhibits the replication of RNA viruses; (2) Viperin also interacts with host proteins involved in innate immune signalling and in metabolic pathways exploited by viruses during their life cycle (e.g. cholesterol biosynthesis). In order to understand the role of fish Viperin in the antiviral response, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the viperin gene has been knocked out by CRISPR/Cas9 genome-editing. The knockout cell line was validated by gDNA sequencing and by western blot upon stimulation with poly(I:C). In order to achieve a global overview of the transcriptional response between the knockout and the wildtype cell lines, we performed a comparative RNA-seq analysis of the whole transcriptome of the two cell lines with or without a 24h-long stimulation with recombinant fathead minnow type I interferon. Our results show that core ISGs are not differentially modulated in the knockout cells compared to their parental counterparts, suggesting that Viperin does not exert a positive feedback on the interferon response in these cells. However, functional enrichment analysis indicates that Viperin might be involved in the regulation of actin skeleton and cholesterol metabolism.
Domaines
Immunité innéeOrigine | Fichiers éditeurs autorisés sur une archive ouverte |
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