Role of Post-transcription RNA Modifications on Zika Virus Gene Expression

项目摘要 寨卡病毒(ZIKV)是一种新出现的蚊媒黄病毒，对人类健康构成巨大威胁 由神经和发育异常以及性传播途径证实。这个 严重的知识差距以及抗病毒疗法和疫苗的缺乏大大增加了。 ZIKV研究的紧迫性。基于N6-甲基腺苷研究的先例 (M6A)在HIV-1、丙型肝炎病毒和ZIKV中，我们假设RNA转录后修饰(PTM)发挥作用 ZIKV在病毒感染中的重要作用--通过调节病毒不同基因表达的基本功能 主持人。了解这些功能将揭示抗病毒开发的新的有希望的靶点。 在各种RNA病毒的基因组上存在PTM已经被知道几十年了。一个 结合免疫沉淀和RNA-SEQ分析的M6A片段联合检测方法 促进了M6A的功能分析。然而，缺乏检测能力严重影响了 阻碍了对140多个其他已知的PTM的这种了解。开发了一种更多功能的 基于质谱学(MS)分析的平台使我们能够研究 PTM存在于模拟和ZIKV感染细胞的总RNA提取液中，以及病毒基因组RNA中 通过亲和捕捉从受感染的细胞和病毒粒子中分离出来。我们令人兴奋的结果表明， ZIKV的基因组由除m6A以外的38种不同类型的PTM装饰，这些令人震惊 随着起源和条件的变化，星座呈现出明显的变化。 在这项建议中，我们将启动病毒PTMS的功能研究，首先研究那些具有 在受感染的细胞和病毒粒子中的独特表达模式。更具体地说，我们将使用MS-Sequence 针对细胞内ZIKV RNA上显著但不显著的不同二甲基胞嘧啶修饰 在包装的RNA上。我们将耗尽安装/移除二甲基胞嘧啶修饰的酶， 突变ZIKV RNA以防止PtM添加，并检查对病毒翻译的生物学影响， 复制和组装。我们将用不同的病毒株和不同的 细胞和蚊子线条。这些结果将为研究PTMS对病毒宿主的影响提供独特的见解 发育过程中的相互作用和基因表达。这些洞察力将为 针对ZIKV感染所必需的宿主因子的新型抗病毒药物的开发，但也可能具有广泛的应用前景 光谱抗病毒药物对所有黄病毒有效。这项研究将有助于确定优先事项和 阐明其余病毒性PTM的框架，该框架将由专门的项目进行 资助金。除了对ZIKV生物学的研究外，这些原理和使能技术 在本R21中开发的将适用于研究涉及RNA故障的其他情况，这将 进一步证实这一项目的广泛变革影响。

PROJECT ABSTRACT Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that presents a formidable health threat substantiated by neurological and developmental anomalies and a sexual transmission route. The significant knowledge gap, as well as the lack of antiviral therapies and vaccines, has greatly increased the urgency of ZIKV research. Based on the precedents set by the investigations of N6-methyladenosine (m6A) in HIV-1, HCV, and ZIKV, we hypothesize that RNA post-transcriptional modifications (PTMs) play important roles in ZIKV infection by regulating essential functions in viral gene expression in different hosts. Understanding these functions will reveal new promising targets for antiviral development. The presence of PTMs on the genome of various RNA viruses has been known for decades. A concerted approach combining immunoprecipitation of m6A-containing fragments and RNA-seq analysis has facilitated functional analysis of m6A. The lack of detection capabilities has however severely hindered such knowledge of the more than 140 other known PTMs. The development of a more versatile platform based on mass spectrometric (MS) analysis has allowed us to examine the global landscape of PTMs present in total RNA extracts of mock- and ZIKV-infected cells, as well as on viral genomic RNA isolated by affinity capture from infected cells and virions. Our exciting results have shown that the genome of ZIKV is decorated by 38 different types of PTMs other than m6A, and that these astonishing constellations present noticeable variations as a function of origin and conditions. In this proposal, we will initiate the functional study of viral PTMs by initially pursuing those with distinctive expression patterns in infected cells and virions. More specifically, we will use MS-sequencing to target different dimethyl-cytosine modifications that were prominent on intracellular ZIKV RNA, but not on packaged RNA. We will deplete the enzymes that install/remove dimethyl-cytosine modifications, mutate the ZIKV RNA to prevent PTM addition, and examine the biological impact on viral translation, replication, and assembly. We will perform these experiments with different viral strains and in different cell and mosquito lines. The results will provide unique insights into the impact of PTMs on virus-host interactions and gene expression during development. These insights will lay the groundwork for the development of novel antivirals targeting host factor essential for ZIKV infection, but also possible broad- spectrum antivirals active across all flaviviruses. This study will help establish the priorities and framework for the elucidation of the remaining viral PTMs, which will be pursued by dedicated project grants. Beyond the investigation of ZIKV biology, these principles and the enabling technologies developed in this R21 will be applicable to study other conditions involving RNA malfunctions, which will further substantiate the broad transformative impact of this project.