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.

FILOVIRES MARBURG病毒（MARV）和埃博拉病毒（EBOV）是阴性的RNA病毒病原体，引起人类严重疾病的周期性暴发。尽管它们作为新兴的病原体和公共卫生威胁的重要性，但Filovirus Biology的许多方面仍被了解。一个主要的知识差距涉及在病毒基因组中存在的非蛋白质编码序列的功能，该序列与病毒mRNA中的5'和3'未翻译区（UTR）相对应。这些序列占MARV基因组的22％和类似数量的EBOV基因组，但几乎没有研究。另一个知识差距涉及将病毒mRNA转化为蛋白质的机制，以及当激活先天免疫反应时如何持续。开始解决这个问题，我们以前在eBov l开放式阅读框架（ORF）的5'Utr中显示了上游开放阅读框（UORF）的作用，该角色在L翻译的调节中。我们进一步暗示了这一UORF，因为它提供了一种在天生抗病毒防御措施下维持L表达的手段。这些数据支持UTR作为翻译调节剂的作用，该调节剂可以在先天抗病毒防御方面调节病毒基因的表达。我们最近对MARV 3'UTR功能的分析表明，核蛋白（NP）mRNA中存在负调节元件，并且L mRNA中存在阳性调节元件。此外，我们先前报道说，MARV基因组似乎是由腺苷脱氨酶活性编辑的，特别是在与核蛋白（NP）mRNA 3'UTR相对应的序列中。我们现在表明，这种突变大大提高了mRNA翻译效率，这表明当IFN诱导的P150形式的腺苷脱氨酶活性在RNA 1（ADAR1）上进行更新时，保持NP表达的一种方法。有趣的是，Wildtype NP 3'Utrs中的调节元素也能够触发先天的免疫调查，但是这种免疫刺激活性可通过假定的ADAR1编辑突变减轻。为了跟进这些观察结果，我们提出了基于翻译和实时病毒实验的组合，以定义MARV和EBOV 3'UTR中的翻译调节元件，并确定ADAR1编辑如何影响病毒基因表达和病毒复制。我们将充分评估MARV和EBOV 5'和3'UTRS触发先天抗病毒反应的能力，从而定义了负责这种归纳的传感器。最后，我们将创建具有野生型和突变体3'UTR序列的重组MARV。这些将在野生型和ADAR1缺陷型细胞和小鼠中进行测试，以确定如何翻译调节元件和ADAR1编辑影响病毒生长和发病机理。累积地，这些研究将为与翻译和先天免疫进化有关的FILOVIRUS-HOST相互作用提供重大的新见解。