Understanding the OAS/RNase L pathway during pathogenic viral infections

项目摘要 核糖核酸酶L（RNase L）是哺乳动物先天性抗病毒反应的关键组分。几十年来，RNase 推测L通过切割核糖体以阻止翻译来减少病毒蛋白质合成。然而，我们和其他人 最近证明，RNase L切割的核糖体是有防御能力的，致病病毒可以 合成蛋白质，尽管激活RNase L。这些观察揭示了知识上的巨大差距 关于RNase L如何发挥作用以及病毒如何逃避它，我们已经证明RNase L几乎可以快速降解， 所有的细胞mRNA。这种活动调节三种细胞过程， 这些研究阐明了致病病毒如何逃避和潜在地劫持RNase L的功能。第一、 RNase L通过降解组成型表达的细胞mRNA将翻译重编程为抗病毒状态， 保留编码抗病毒蛋白的宿主mRNA（例如，I型干扰素），其允许抗病毒蛋白质合成。 重要的是，由几种致病病毒编码的mRNA（例如，登革病毒）同样逃避RNase L介导的 mRNA衰变，从而允许病毒蛋白质合成。这一观察阐明了致病病毒如何合成 蛋白质，尽管激活RNase L。本申请提出表征RNase L介导的mRNA衰变 第二，RNase L激活触发核转录抑制， mRNA输出。这是一个关键的抗病毒机制，拮抗甲型流感病毒蛋白合成，但它也 下调宿主抗病毒蛋白的表达（例如，I型干扰素）。重要的是，致病性病毒（例如， 登革病毒）激活这种RNase L依赖性途径，导致宿主抗病毒mRNA被隔离在 原子核这一观察结果表明，病毒可能会劫持RNase L的这种功能，以限制宿主的抗病毒蛋白 生产该应用旨在确定RNase L如何抑制mRNA输出，其拮抗的病毒的广度， 在致病性病毒感染期间它如何影响宿主抗病毒基因表达。第三，RNase L调节组装 细胞质抗病毒核糖核蛋白复合物。具体而言，RNase L抑制应激颗粒的组装， 促进称为RNase L依赖体的替代应力颗粒样核糖核蛋白复合物的组装。 RNase L依赖体是SARS-CoV-2或登革病毒应答中组装的主要抗病毒颗粒 感染，但其功能完全未知。本申请旨在确定抗病毒应激的功能 颗粒和RNase L依赖性机构，并确定如何调节RNase L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细胞周期的理解。 OAS/RNase L途径、先天免疫抗病毒基因诱导和病毒学。此外，它将促进一般 通过广泛表征与非感染性疾病相关的基本细胞、分子和RNA生物学， 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RNase L生物学的有前景的抗病毒、免疫调节和抗癌疗法的开发。

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.