Understanding the OAS/RNase L pathway during pathogenic viral infections

项目总结 核糖核酸酶L(核糖核酸酶L)是哺乳动物先天抗病毒反应的重要组成部分。几十年来，RNase L被推测通过裂解核糖体来阻止翻译来减少病毒蛋白质的合成。然而，我们和其他人 最近证明，核糖核酸酶L裂解的核糖体具有翻译能力，并且致病病毒可以 在激活核糖核酸酶的情况下合成蛋白质。这些观察揭示了知识上的重大差距。 关于核糖核酸酶L是如何工作的，以及病毒是如何规避它的。我们已经证明，核糖核酸酶L几乎迅速降解 激活时所有细胞内的mRNAs。这一活动调节了三个细胞过程，扩展了我们的理解 阐明了致病病毒如何逃避并可能劫持核糖核酸酶L的功能。第一, 核糖核酸酶L通过降解结构性表达的细胞mRNA将翻译重新编程为抗病毒状态，而同时 保留编码抗病毒蛋白(例如，I型干扰素)的宿主mRNAs，这允许抗病毒蛋白的合成。 重要的是，由几种致病病毒(如登革热病毒)编码的mRNAs类似地避开了核糖核酸酶L介导的 信使核糖核酸腐烂，从而允许病毒蛋白质的合成。这一观察阐明了致病病毒是如何合成的 蛋白质激活核糖核酸酶L这一应用建议表征核糖核酸酶L介导的mR NA衰变 途径，并确定宿主和病毒的mRNAs如何逃避它。第二，核糖核酸酶L激活触发核抑制 信使核糖核酸输出。这是对抗甲型流感病毒蛋白质合成的关键抗病毒机制，但它也 下调宿主抗病毒蛋白(如I型干扰素)的表达。重要的是，致病病毒(例如， 登革病毒)激活这一依赖于L的核糖核酸酶途径，导致宿主抗病毒mRNAs在 原子核。这一观察结果表明，病毒可能劫持了核糖核酸酶L的这一功能，以限制宿主抗病毒蛋白 制作。这项应用旨在确定核糖核酸酶L是如何抑制基因输出的，它对抗的病毒的广度， 在病原性病毒感染期间，它如何影响宿主抗病毒基因的表达。第三，网易L规范集结 细胞质抗病毒核糖核蛋白复合体。具体地说，核糖核酸酶L抑制应激颗粒和 促进另一种名为核糖核酸酶L依赖体的应激颗粒状核糖核蛋白复合体的组装。 核糖核酸酶L依赖的小体是为应对SARS-CoV-2或登革病毒而组装的主要抗病毒颗粒 感染，但它们的功能完全未知。这项应用旨在确定抗病毒应激的功能 目的是研究核糖核酸酶L对颗粒和核糖核酸酶L依赖小体的调节作用，并确定核糖核酸酶L对它们的调节如何改变抗病毒反应。 了解这些细胞过程的机制和功能将促进我们对 Oas/核糖核酸酶L通路；天然免疫抗病毒基因诱导；病毒学。此外，它还将促进一般 通过广泛描述与非传染性相关的基本细胞、分子和RNA生物学来进行医学研究 疾病，包括自身免疫性疾病、神经变性和癌症。最后，拟议的研究将支持 基于核糖核酸酶L生物的抗病毒、免疫调节和抗癌治疗的研究进展。

PROJECT SUMMARY Ribonuclease L (RNase L) is a key component of the mammalian innate antiviral response. For decades, RNase L was presumed to reduce viral protein synthesis by cleaving ribosomes to arrest translation. However, we and others recently demonstrated that RNase L-cleaved ribosomes are translation-competent, and that pathogenic viruses can synthesize proteins despite activating RNase L. These observations have revealed a significant gap in knowledge regarding how RNase L functions and how viruses evade it. We have demonstrated that RNase L rapidly degrades nearly all cellular mRNAs upon activation. This activity regulates three cellular processes that have expanded our understanding of RNase L and that have elucidated how pathogenic viruses evade and potentially hijack RNase L functions. First, RNase L reprograms translation to an antiviral state by degrading constitutively expressed cellular mRNAs while sparing host mRNAs encoding antiviral proteins (e.g., type I interferons), which permits antiviral protein synthesis. Importantly, the mRNAs encoded by several pathogenic viruses (e.g., dengue virus) similarly evade RNase L-mediated mRNA decay, thus permitting viral protein synthesis. This observation has elucidated how pathogenic viruses synthesize proteins despite activating RNase L. This application proposes to characterize the RNase L-mediated mRNA decay pathway and determine how host and viral mRNAs evade it. Second, RNase L activation triggers the inhibition of nuclear mRNA export. This is a critical antiviral mechanism that antagonizes influenza A virus protein synthesis, but it also downregulates the expression of host antiviral proteins (e.g., type I interferons). Importantly, pathogenic viruses (e.g., dengue virus) activate this RNase L-dependent pathway, resulting in sequestration of host antiviral mRNAs in the nucleus. This observation suggests that viruses potentially hijack this function of RNase L to limit host antiviral protein production. This application aims to determine how RNase L inhibits mRNA export, the breadth of viruses it antagonizes, how it impacts host antiviral gene expression during pathogenic viral infections. Third, RNase L regulates the assembly of cytoplasmic antiviral ribonucleoprotein complexes. Specifically, RNase L inhibits the assembly of stress granules and promotes the assembly of an alternative stress granule-like ribonucleoprotein complex termed RNase L-dependent body. RNase L-dependent bodies are the predominant antiviral granule assembled in response to SARS-CoV-2 or dengue virus infection, yet their function is completely unknown. This application aims to determine the function of antiviral stress granules and RNase L-dependent bodies and to determine how their regulation by RNase L alters the antiviral response. Understanding the mechanisms and functions of these cellular processes will advance our understanding of the OAS/RNase L pathway, innate immune antiviral gene induction, and virology. Moreover, it will promote general medicine by broadly characterizing fundamental cellular, molecular, and RNA biology that is relevant to non-infectious diseases, including autoimmune diseases, neurodegeneration, and cancer. Lastly, the proposed research will support the development of promising antiviral, immunomodulatory, and anticancer therapies based on RNase L biology.