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

• 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
• 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' Untranslated Regions; 5' 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

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(Marburg Virus，MARV)和埃博拉病毒(Ebola Virus，EBOV)是引起人类周期性严重疾病爆发的负义RNA病毒病原体。尽管丝状病毒作为新出现的病原体和公共卫生威胁很重要，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编码序列的功能(S)，这些非蛋白编码序列对应于病毒mRNA5‘和3’非翻译区(UTRs)。这些序列包括约22%的Marv基因组和类似数量的EBOV基因组，但几乎没有人研究过它们。另一个知识空白涉及病毒mRNAs被翻译成蛋白质的机制，以及当先天免疫反应被激活时这一机制如何维持。为了解决这个问题，我们先前证明了上游开放阅读框架在调控L翻译中的作用，该框架存在于EBOV L开放阅读框架的5‘UTR中。我们进一步暗示，该uORF为在天然抗病毒防御被激活的条件下维持L的表达提供了一种手段。这些数据支持UTRs作为翻译调节器的作用，它可以在面对固有的抗病毒防御时调节病毒基因的表达。我们最近对MARV3‘非编码区功能的研究表明，在核蛋白(NP)基因中存在负调控元件，在L基因中存在正调控元件。此外，我们以前报道过Marv基因组似乎由腺苷脱氨酶活性编辑，特别是在与核蛋白(NP)mRNA3‘UTR相对应的序列中。我们现在表明，这种突变大大提高了mRNA的翻译效率，这表明当干扰素诱导的作用于RNA 1的腺苷脱氨酶P150形式(ADAR1)上调时，可以维持NP的表达。有趣的是，野生型NP 3‘UTR中的调节元件也能够触发先天性免疫反应，但这种免疫刺激活性被假定的ADAR1编辑突变所缓解。为了跟进这些观察到的结果，我们提出了一项结合转基因和活病毒的实验，以定义Marv和EBOV 3‘UTRs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，确定负责这种诱导的传感器。最后，我们将创建带有野生型和突变型3‘非编码区序列的重组MARV。这些将在野生型和ADAR1缺陷的细胞和小鼠中进行测试，以确定翻译调节元件和ADAR1编辑如何影响病毒的生长和发病。总而言之，这些研究将为丝状病毒-宿主相互作用提供重要的新见解，这些相互作用与翻译和先天免疫逃避有关。