Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.

IF 3.6 3区 医学 Q2 IMMUNOLOGY
Lise Chaumont, Mathilde Peruzzi, François Huetz, Claudine Raffy, Jérôme Le Hir, Jules Minke, Pierre Boudinot, Bertrand Collet
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Abstract

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.

鲑鱼双链 RNA 依赖性蛋白激酶激活细胞凋亡并抑制蛋白质合成
dsRNA依赖性蛋白激酶R(PKR)是先天性免疫的一个关键因素。它参与了翻译抑制、细胞凋亡以及促炎和 IFN 反应的增强。然而,这些抗病毒功能是如何在进化过程中保持不变的,在很大程度上仍是未知数。为了评估鲑鱼 PKR 在抗病毒反应中的作用,我们在大鳞鲑(Oncorhynchus tshawytscha)细胞系中进行了过表达和基因敲除研究。研究人员克隆了来自独特 pkr 基因的三种不同 mRNA 异构体,分别命名为 pkr-fl(全长)、pkr-ml(中长)和 pkr-sl(短),并利用 CRISPR/Cas9 基因组编辑技术开发了 pkr-/- 克隆鱼细胞系。PKR-FL 包括一个 N 端 dsRNA 结合结构域和一个 C 端激酶结构域,而 PKR-ML 和 PKR-SL 则分别显示了一个截短的激酶结构域或没有激酶结构域。PKR-FL 在 IFNA2 刺激时被诱导,但在病毒性出血性败血症病毒(VHSV)感染时则不被诱导。过表达实验表明,只有 PKR-FL 具有抗病毒功能,包括激活细胞凋亡和抑制新蛋白质合成。基因敲除实验证实,PKR 参与了 VHSV 感染后期的细胞凋亡激活。内源性 PKR 在 IFNA2 处理后的 poly(I:C) 转染翻译抑制中也起着关键作用。但它并不参与 VHSV 感染过程中的翻译抑制。细胞外和细胞内滴定显示,内源性 PKR 并不直接抑制病毒复制,但显然有利于病毒释放到上清液中,很可能是通过触发晚期细胞凋亡。总之,我们的数据证实了鲑鱼的 PKR 具有保守的分子功能,而 VHSV 似乎通过颠覆策略绕过了这些功能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of immunology
Journal of immunology 医学-免疫学
CiteScore
8.20
自引率
2.30%
发文量
495
审稿时长
1 months
期刊介绍: The JI publishes novel, peer-reviewed findings in all areas of experimental immunology, including innate and adaptive immunity, inflammation, host defense, clinical immunology, autoimmunity and more. Special sections include Cutting Edge articles, Brief Reviews and Pillars of Immunology. The JI is published by The American Association of Immunologists (AAI)
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