An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.
An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.
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DOI：
10.1371/journal.pbio.1002364
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发表时间：
2016-03
期刊：
影响因子：
9.8
通讯作者：
Ghazal P
中科院分区：
文献类型：
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作者：
Robertson KA;Hsieh WY;Forster T;Blanc M;Lu H;Crick PJ;Yutuc E;Watterson S;Martin K;Griffiths SJ;Enright AJ;Yamamoto M;Pradeepa MM;Lennox KA;Behlke MA;Talbot S;Haas J;Dölken L;Griffiths WJ;Wang Y;Angulo A;Ghazal P
在无脊椎动物中，小干扰RNA是细胞自主抗病毒免疫的先锋。相反，由干扰素（IFN）信号传导启动的抗病毒机制在哺乳动物中占主导地位。虽然已知哺乳动物IFN诱导的miRNA抑制特定病毒，但不知道IFN信号传导下游的宿主定向的microRNA是否在介导广泛的抗病毒抗性中起作用。通过利用IFN-活化的巨噬细胞中新转录的RNA和pri/pre-miRNA的4-硫代尿苷标记进行RNA周转的综合、系统、全局分析，我们鉴定了由miR-342- 5 p介导的新的转录后病毒防御机制。在ChIP和定点启动子突变实验的基础上，我们发现miR-342- 5 p的合成通过IFN诱导的转录因子IRF 1与抗病毒IFN反应偶联。引人注目的是，我们发现miR-342- 5 p使用多击机制在不同的功能水平上抑制途径来靶向甲羟戊酸-甾醇生物合成：转录通过SREBF 2，转录后通过miR-33，以及酶促通过IDI 1和SC 4 MOL。基于质谱的脂质组学和酶测定证明靶向机制减少巨噬细胞中的中间固醇途径代谢物和总胆固醇。这些结果揭示了一个以前未被认识的机制，IFN调节甾醇途径。已知固醇途径是巨噬细胞IFN抗病毒反应的组成部分，并且我们表明miR-342- 5 p对多种不相关的病原性病毒如巨细胞病毒和甲型流感（H1N1）发挥广泛的抗病毒作用。代谢拯救实验证实了这些效应的特异性，并证明了不相关的病毒对其复制具有不同的甲羟戊酸和甾醇途径要求。因此，本研究提出了广泛的抗病毒防御的一般概念，通过一个单一的宿主途径的多击靶向。干扰素诱导的miRNA通过多个靶点抑制固醇生物合成途径，从而帮助建立对不相关的临床重要病毒的广泛细胞抗性。感染细胞在感染后的最初几分钟到几小时内对病毒的反应可以决定疾病是否发展并影响宿主的长期生存。在哺乳动物中，与使用小RNA对抗病毒感染的植物和苍蝇不同，病毒诱导的干扰素反应是一个关键的早期事件，导致广泛的抗感染保护。干扰素是一种分泌的宿主蛋白，它与感染和未感染细胞表面的受体结合，并激活生物化学途径，深刻改变数百种细胞基因的表达，包括编码microRNA的基因。这些基因中只有少数基因的抗病毒功能是已知的，并且不知道大多数基因如何有助于广泛地保护免受许多不同病毒的侵害。在这项研究中，我们发现了一种干扰素调节的microRNA（miR-342- 5 p），它通过胆固醇生物合成途径促进了广泛的宿主细胞对感染的免疫。我们发现，miR-342- 5 p通过多击策略来实现这一点，下调固醇生物合成的主要调节因子以及途径中的几种特异性靶向酶。多种病毒的复制依赖于甾醇生物合成途径的许多代谢产物分支。值得注意的是，我们的研究表明，通过在单一途径中利用关键分支点的多点靶向，miR-342- 5 p能够抑制从疱疹到流感病毒等不相关的临床重要病原体的复制。
In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. An interferon-induced miRNA suppresses the sterol biosynthesis pathway via multiple targets, thereby helping establish broad cellular resistance to unrelated clinically significant viruses. How infected cells respond to a virus during the first minutes to hours after infection can determine whether a disease develops and influences the host’s long-term survival. In mammals, unlike plants and flies that use small RNAs to fight viral infections, virus-induced interferon responses are a critical early event resulting in broad protection against infection. Interferon is a secreted host protein that binds to receptors on the surface of infected and uninfected cells and activates biochemical pathways that profoundly change the expression of hundreds of cellular genes, including those encoding microRNAs. The antiviral functions of only a handful of these genes are understood, and it is not known how the majority contribute to broadly protect against many different viruses. In this study, we uncover an interferon-regulated microRNA (miR-342-5p) that contributes to broad host cell immunity against infection through the cholesterol biosynthesis pathway. We show that miR-342-5p does this through a multihit strategy, turning down the master regulator of sterol biosynthesis as well as several specifically targeted enzymes within the pathway. A wide range of viruses depend on a number of the metabolite side-branches of the sterol biosynthesis pathway for their replication. Notably, our study reveals that by utilising multihit targeting of key branch-points in a single pathway, miR-342-5p is able to inhibit the replication of unrelated, clinically significant pathogens ranging from Herpes to Flu viruses.