Effect of m6A editing of RNA on influenza A virus replication

虽然DNA和蛋白质的化学修饰的重要性是众所周知的，但对mRNA转录物的转录后修饰如何影响其功能知之甚少。在哺乳动物细胞mRNA上看到的最常见的修饰碱基是N6-甲基腺苷（m6 A），最近的数据表明，m6 A形成的完全丧失可能具有严重的有害后果，例如，阻断多能干细胞的分化。然而，m6 A如何调节mRNA表达仍有待确定。此外，先前的研究表明，包括甲型流感病毒（IAV）在内的几种致病性人类病毒编码的RNA会进行m6 A编辑，这表明这种修饰可能对病毒复制产生积极影响。事实上，来自该实验室和其他实验室的最新数据表明，m6 A编辑可以显着增强培养的T细胞中的HIV-1基因表达和复制。这项拨款提案旨在充分定义IAV转录本上m6 A修饰的位点，并测试m6 A编辑增强IAV基因表达并因此复制的假设。使用PAR-CLIP技术，我们已经在IAV基因组上的病毒mRNA/cRNA和vRNA转录物上鉴定了人YTHDF m6 A阅读器蛋白的几个特异性结合位点。与m6 A编辑可以正向调节IAV基因表达的假设一致，我们已经观察到，在人A549细胞中，人YTHDF 2阅读蛋白的过表达，而不是相关的YTHDF 1蛋白的过表达，极大地增强了IAV的复制。这项资助的目的首先是在单核苷酸分辨率下确定IAV转录本上m6 A修饰的精确位置，然后量化每个位点的编辑程度。然后，我们将使用靶向诱变来精确地确定IAV基因组不同片段上的特定m6 A修饰如何影响IAV基因表达和复制。同时，我们将研究参与“写入”、“阅读”和“擦除”m6 A标记的关键细胞蛋白的过表达，或通过使用CRISPR/Cas进行基因组编辑消除它们，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m6 A编辑如何影响IAV复制，并可能导致新的方法来增强培养物中的IAV复制，例如用于IAV疫苗生产，或抑制体内IAV复制，例如通过瞬时抑制m6 A编辑。

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.