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

丝状病毒马尔堡病毒（MARV）和埃博拉病毒（EBOV）是引起人类严重疾病周期性暴发的负义RNA病毒病原体。尽管它们作为新兴病原体和公共卫生威胁具有重要意义，但线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对应于病毒mrna中5 ‘和3 ’非翻译区（utr）的非蛋白编码序列的功能。这些序列包含约22%的MARV基因组和相似数量的EBOV基因组，但几乎没有被研究过。另一个知识缺口涉及病毒mrna转化为蛋白质的机制，以及当先天免疫反应被激活时，这种机制如何维持。为了解决这个问题，我们之前证明了上游开放阅读框（uORF）在调节L翻译中的作用，它存在于EBOV L开放阅读框（ORF）的5'UTR中。我们进一步暗示，在先天抗病毒防御被激活的情况下，这种uORF提供了一种维持L表达的手段。这些数据支持utr作为翻译调节因子的作用，它可以在面对先天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，而L mRNA中存在正调控元件。此外，我们之前报道了MARV基因组似乎被腺苷脱氨酶活性编辑，特别是在与核蛋白（NP） mRNA 3'UTR对应的序列中。我们现在发现，这种突变大大提高了mRNA的翻译效率，表明当ifn诱导的作用于RNA 1的p150形式的腺苷脱氨酶（ADAR1）上调时，有一种维持NP表达的方法。有趣的是，野生型NP 3'UTR中的调控元件也能够触发先天免疫反应，但这种免疫刺激活性被假定的ADAR1编辑突变减轻。为了跟进这些观察结果，我们提出了一种基于转染和活病毒的结合实验，以确定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复制。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，并定义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型3'UTR序列的重组marv。这些将在野生型和ADAR1缺陷细胞和小鼠中进行测试，以确定翻译调节元件和ADAR1编辑如何影响病毒生长和发病机制。总的来说，这些研究将为丝状病毒与宿主相互作用的翻译和先天免疫逃避提供重要的新见解。

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.