Effect of m6A editing of RNA on influenza A virus replication

RNA m6A 编辑对甲型流感病毒复制的影响

• 批准号: 9296268
• 负责人: BRYAN R. CULLEN
• 金额: $ 23.85万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-06 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9296268
• 关键词: A549; Adenosine; Affect; Antibodies; Applications Grants; Binding Sites; Cell Nucleus; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Cultured Cells; DNA Modification Process; Data; Development; Embryo; Gene Expression; Genes; Genetic Transcription; Grant; HIV-1; Hemagglutinin; Human; Individual; Influenza A virus; Laboratories; Lead; Location; Mammals; Maps; Mediating; Messenger RNA; Methylation; Modification; Mutagenesis; Mutate; Nucleotides; Organism; Pathogenicity; Pathway interactions; Plants; Play; Pluripotent Stem Cells; Positioning Attribute; Post-Translational Protein Processing; Proteins; RNA Editing; RNA Interference; Reader; Reading; Reporting; Resolution; Role; Site; Somatic Cell; Structure; T-Lymphocyte; Techniques; Technology; Testing; Transcript; Translations; Ursidae Family; Vaccine Production; Viral; Viral Genes; Viral Genome; Virus; Virus Diseases; Virus Replication; Work; Writing; base; egg; genome editing; in vivo; knock-down; mRNA Expression; methyl group; novel strategies; overexpression; pathogen; viral RNA

While the importance of chemical modifications of DNA and proteins is well established, relatively little is known about how the post-transcriptional modification of mRNA transcripts affects their function. The most common modified base seen on cellular mRNAs in mammals is N6-methyladenosine (m6A), and recent data demonstrate that the total loss of m6A formation can have severe deleterious consequences, for example, blocking the differentiation of pluripotent stem cells. However, how m6A regulates mRNA expression remains to be established. Moreover, previous work has revealed that several pathogenic human viruses, including influenza A virus (IAV), encode RNAs that undergo m6A editing, suggesting that such modifications might exert a positive effect on virus replication. Indeed, recent data from this laboratory, and others, demonstrate that m6A editing can significantly enhance HIV-1 gene expression and replication in cultured T cells. This grant proposal seeks to fully define the sites of m6A modification on IAV transcripts and to test the hypothesis that m6A editing enhances IAV gene expression and, hence, replication. Using the PAR-CLIP technique, we have identified several specific binding sites for the human YTHDF m6A reader proteins on the IAV genome on both the viral mRNA/cRNA and vRNA transcripts. Consistent with the hypothesis that m6A editing can positively regulate IAV gene expression, we have observed that overexpression of the human YTHDF2 reader protein, but not of the related YTHDF1 protein, in human A549 cells greatly enhances IAV replication. This grant first aims to define the precise locations of m6A modifications on IAV transcripts at single nucleotide resolution and to then quantify the degree of editing at each site. We will then use targeted mutagenesis to determine precisely how specific m6A modifications on different segments of the IAV genome affect IAV gene expression and replication. In parallel, we will examine how the overexpression of key cellular proteins involved in “writing”, “reading” and “erasing” m6A marks, or their elimination by genome editing using CRISPR/Cas, affects IAV gene expression and replication in A549 cells. Together, these data will reveal how m6A editing affects IAV replication and may lead to new approaches to the enhancement of IAV replication in culture, for use for example in IAV vaccine production, or the inhibition of IAV replication in vivo, for example by transient inhibition of m6A editing.

虽然 DNA 和蛋白质化学修饰的重要性已广为人知，但对于 mRNA 转录物的转录后修饰如何影响其功能却知之甚少。哺乳动物细胞 mRNA 上最常见的修饰碱基是 N6-甲基腺苷 (m6A)，最近的数据表明，m6A 形成的完全丧失可能会产生严重的有害后果，例如，阻碍多能干细胞的分化。然而，m6A 如何调节 mRNA 表达仍有待确定。此外，之前的工作表明，包括甲型流感病毒（IAV）在内的几种致病性人类病毒编码经过m6A编辑的RNA，表明这种修饰可能对病毒复制产生积极影响。事实上，该实验室和其他实验室的最新数据表明，m6A 编辑可以显着增强培养 T 细胞中的 HIV-1 基因表达和复制。该资助提案旨在全面定义 IAV 转录本上 m6A 修饰的位点，并测试 m6A 编辑增强 IAV 基因表达并因此增强复制的假设。使用 PAR-CLIP 技术，我们在病毒 mRNA/cRNA 和 vRNA 转录物上鉴定了 IAV 基因组上人类 YTHDF m6A 阅读器蛋白的几个特异性结合位点。与 m6A 编辑可以正向调节 IAV 基因表达的假设一致，我们观察到，在人 A549 细胞中，人 YTHDF2 阅读器蛋白（而非相关 YTHDF1 蛋白）的过表达极大地增强了 IAV 复制。该资助首先旨在以单核苷酸分辨率定义 IAV 转录本上 m6A 修饰的精确位置，然后量化每个位点的编辑程度。然后，我们将使用定向诱变来精确确定 IAV 基因组不同片段上的特定 m6A 修饰如何影响 IAV 基因表达和复制。与此同时，我们将研究参与“写入”、“读取”和“擦除”m6A 标记的关键细胞蛋白的过度表达，或通过使用 CRISPR/Cas 进行基因组编辑来消除它们，如何影响 A549 细胞中 IAV 基因的表达和复制。总之，这些数据将揭示 m6A 编辑如何影响 IAV 复制，并可能带来增强 IAV 在培养物中复制的新方法，例如用于 IAV 疫苗生产，或抑制 IAV 体内复制，例如通过瞬时抑制 m6A 编辑。