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: 10593400
• 负责人: Christopher F Basler
• 金额: $ 40.76万
• 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/10593400
• 关键词: Intersection; Between; Viral; Translation; Innate

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）是在人类中引起严重疾病的周期性爆发的负义RNA病毒病原体。尽管它们作为新兴病原体和公共卫生威胁的重要性，但丝状病毒生物学的许多方面仍未得到充分研究。一个主要的知识缺口涉及丝状病毒基因组中存在的非蛋白质编码序列的功能，所述非蛋白质编码序列对应于病毒mRNA中的5'和3'非翻译区（UTR）。这些序列包含约22%的MARV基因组和类似量的EBOV基因组，但几乎没有研究。另一个知识缺口涉及病毒mRNA被翻译成蛋白质的机制，以及当先天免疫反应被激活时，这种机制如何持续。为了解决这个问题，我们先前证明了存在于EBOV L开放阅读框（ORF）的5 'UTR中的上游开放阅读框（uORF）在L翻译调节中的作用。我们进一步暗示，这个uORF提供了一种手段，以维持L表达的条件下，先天性抗病毒防御被激活。这些数据支持UTR作为翻译调节因子的作用，其可以在面对先天性抗病毒防御时调节病毒基因表达。我们最近对MARV 3 'UTR功能的分析表明，核蛋白（NP）mRNA中存在负调控元件，L mRNA中存在正调控元件。此外，我们以前报道过MARV基因组似乎是由腺苷脱氨酶活性编辑的，特别是在对应于核蛋白（NP）mRNA 3 'UTR的序列中。我们现在表明，这样的突变大大增加mRNA的翻译效率，这表明一种手段，以维持NP表达时，IFN诱导的p150形式的腺苷脱氨酶作用于RNA 1（ADAR 1）上调。有趣的是，野生型NP 3 'UTR内的调控元件也能够触发先天免疫应答，但这种免疫刺激活性被推定的ADAR 1编辑突变所减轻。为了跟进这些观察结果，我们提出了基于转染和活病毒的实验的组合，以定义MARV和EBOV 3'UTR中的翻译调控元件，并确定ADAR 1编辑如何影响病毒基因表达和病毒复制。我们将全面评估MARV和EBOV 5'和3' UTR触发先天性抗病毒反应的能力，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ADAR 1缺陷细胞和小鼠中进行测试，以确定翻译调控元件和ADAR 1编辑如何影响病毒生长和发病机制。累积起来，这些研究将为与翻译和先天免疫逃避相关的丝状病毒-宿主相互作用提供重要的新见解。