Intersection Between Viral Translation and Innate Immunity in the Context of Filovirus Infection

丝状病毒感染背景下病毒翻译与先天免疫之间的交叉

基本信息

批准号：
10425317
负责人：
Christopher F Basler
金额：
$ 61.95万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-13 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10425317
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Address Adenosine Africa Angola Antiviral Response Biological Assay Biological Models Biology Case Fatality Rates Cell Culture Techniques Cells Cessation of life Code Containment Data Detection Disease Disease Outbreaks Double-Stranded RNA EIF-2alpha Ebola virus Elements Enzymes Family Fatality rate Filoviridae Infections Filovirus Gene Expression Genes Genetic Translation Genome Growth Guanosine Health Homeostasis Hot Spot Hour Human Immune Immune Evasion Immune response Immune signaling In Vitro Infection Innate Immune Response Inosine Interferons Knock-out Knockout Mice Knowledge Laboratories Life Cycle Stages Marburgvirus Messenger RNA MicroRNAs Modeling Molecular Biology Mus Mutation Natural Immunity Nucleoproteins Open Reading Frames Pathogenesis Pattern recognition receptor Peptide Initiation Factors Periodicity Phosphorylation Phosphotransferases Protein Isoforms Proteins Public Health RNA RNA Viruses Recombinants Regulation Regulatory Element Reporting Research Role Site Testing Transfection Translating Translation Initiation Translations Untranslated Regions Viral Viral Genes Viral Genome Viral Physiology Virus Virus Replication Wild Type Mouse Work Zoonoses adenosine deaminase base emerging pathogen experimental study follow-up in vivo insight interest mutant pathogen pathogenic virus response reverse genetics sensor viral fitness

项目摘要

The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) are negative-sense RNA viral pathogens that cause periodic outbreaks of severe disease in humans. Despite their importance as emerging pathogens and as public health threats, many aspects of filovirus biology remain understudied. One major knowledge gap concerns the function(s) of the non-protein coding sequences present in filovirus genomes that correspond to 5’ and 3’ untranslated regions (UTRs) in viral mRNAs. These sequences comprise ~22% of the MARV genome and a similar amount of the EBOV genome and yet have been barely studied. Another knowledge gap concerns mechanisms by which viral mRNAs are translated to protein and how this may be sustained when innate immune responses are activated. Beginning to address this, we previously demonstrated a role for an upstream open reading frame (uORF), present in the 5’UTR of the EBOV L open reading frame (ORF), in the regulation of L translation. We further implicated this uORF as providing a means to sustain L expression under conditions innate antiviral defenses are activated. These data support a role for the UTRs as translational regulators which can modulate viral gene expression in the face of innate antiviral defenses. Our recent profiling of MARV 3’UTR function suggests negative-regulatory elements are present in the nucleoprotein (NP) mRNA and that positive regulatory elements are present in the L mRNA. In addition, we previously reported that the MARV genome appears to be edited by adenosine deaminase activity, particularly in sequences corresponding to the nucleoprotein (NP) mRNA 3’UTR. We now show that such mutations substantially increase mRNA translation efficiency, suggesting a means to maintain NP expression when the IFN-induced p150 form of adenosine deaminase acting on RNA 1 (ADAR1) is upregulated. Interestingly, the regulatory elements within the wildtype NP 3’UTR are also capable of triggering innate immune responses, but this immune stimulating activity is alleviated by the presumptive ADAR1 editing mutations. To follow up on these observations, we propose a combination of transfection- and live virus-based experiments to define the translation regulatory elements in MARV and EBOV 3’ UTRs and to determine how ADAR1 editing impacts viral gene expression and virus replication. We will fully evaluate the capacity of MARV and EBOV 5’ and 3’UTRs to trigger innate antiviral responses, defining the sensors responsible for this induction. Finally, we will create recombinant MARVs with wildtype and mutant 3’UTR sequences. These will be tested in wildtype and ADAR1-deficient cells and mice in order to determine how translational regulatory elements and ADAR1 editing impact viral growth and pathogenesis. Cumulatively, these studies will provide significant new insight into filovirus-host interactions related to translation and innate immune evasion.

丝状病毒马尔堡病毒 (MARV) 和埃博拉病毒 (EBOV) 是负义 RNA 病毒病原体，会导致人类周期性爆发严重疾病，尽管它们作为新兴病原体和公共卫生威胁具有重要意义，但丝状病毒生物学的许多方面仍未得到充分研究。一个主要的知识空白涉及丝状病毒基因组中存在的非蛋白质编码序列的功能，这些序列对应于病毒 mRNA 中的 5' 和 3' 非翻译区 (UTR)。序列包含约 22% 的 MARV 基因组和相似数量的 EBOV 基因组，但几乎没有研究过病毒 mRNA 翻译成蛋白质的机制以及当先天免疫反应被激活时如何维持这种机制。为了解决这个问题，我们之前证明了存在于 EBOV L 开放阅读框 (ORF) 5'UTR 中的上游开放阅读框 (uORF) 在 L 翻译调节中的作用。提供了一种在先天抗病毒防御激活的情况下维持 L 表达的方法。这些数据支持 UTR 作为翻译调节因子的作用，可以在先天抗病毒防御的情况下调节病毒基因表达。我们最近对 MARV 3'UTR 功能的分析表明。负调控元件存在于核蛋白 (NP) mRNA 中，正调控元件存在于 L mRNA 中。此外，我们之前报道过 MARV 基因组似乎是由腺苷编辑的。脱氨酶活性，特别是在核蛋白 (NP) mRNA 3'UTR 的相应序列中，我们现在表明，此类突变显着提高了 mRNA 翻译效率，表明当 IFN 诱导的 p150 形式的腺苷脱氨酶作用于 RNA 时，有一种维持 NP 表达的方法。 1 (ADAR1) 上调，野生型 NP 3'UTR 内的调节元件也能够触发先天免疫反应，但这种免疫刺激活性会被 NP 3'UTR 减弱。为了跟进这些观察结果，我们建议结合基于转染和活病毒的实验来定义 MARV 和 EBOV 3' UTR 中的翻译调控元件，并确定 ADAR1 编辑如何影响病毒基因表达和病毒。我们将全面评估 MARV 和 EBOV 5' 和 3'UTR 触发先天抗病毒反应的能力，定义负责这种诱导的传感器最后，我们将创建重组体。具有野生型和突变型 3'UTR 序列的 MARV 将在野生型和 ADAR1 缺陷细胞和小鼠中进行测试，以确定翻译调控元件和 ADAR1 编辑如何影响病毒生长和发病机制。与翻译和先天免疫逃避相关的丝状病毒-宿主相互作用。