Epitranscriptomic modification of HIV-1 transcripts: Effects of drugs of abuse

摘要 虽然病毒RNA功能的表观转录组调控已经开始引起相当大的关注， 到目前为止，这项工作完全集中在单一的表转录组修饰上，即，添加甲基 腺苷（m6 A）的N6位。这种关注是可以理解的，因为m6 A是最常见的 细胞mRNA的表观转录组修饰，并且已知约40年来病毒转录物也 含有大量的m6 A。此外，我们和其他人已经报道了m6 A调节HIV-1复制， 最近，我扩展了这项工作，表明m6 A也增强了复制和致病性， 甲型流感病毒（IAV）。第二个表观修饰，尿苷异构化为假尿苷 （1），以前被认为是局限于非编码RNA，最近已被证明是几乎一样普遍， 我们现在已经观察到，包装成病毒体的基因组RNA（gRNA） 的逆转录病毒鼠白血病病毒显示出一系列的表位转录组修饰，不仅包括m6 A 以及5-甲基胞嘧啶（5 mC）残基。在这份拨款申请中，我们建议使用超高 高效液相色谱串联质谱法（UPLC-MS/MS），以系统地鉴定和 定量从HIV-1分离的高度纯化gRNA上存在的表位转录组修饰 病毒粒子然后，我们将绘制三个最普遍的epitranscriptomic修改的精确位置， 在CD 4 + T细胞和骨髓细胞中表达的gRNA和HIV-1 mRNA。接下来，我们将默默地 在感染性HIV-1前病毒的背景下突变这些修饰的位点，并确定这些位点是否以及如何突变。 修饰影响病毒基因表达和复制。我们还将寻求确定细胞因子， 存款这些epitranscriptomic标记的HIV-1转录，然后我们将测试的影响，表达损失， 或过表达，这些因子对HIV-1在CD 4 + T细胞中复制的影响。最后，我们将分析 环境压力，如热休克和滥用药物治疗，特别是尼古丁和 吗啡对HIV-1中表达的细胞和病毒转录物的表位转录组修饰水平的影响 被感染的细胞同时，我们还将确定这些相同的处理是否影响了 增加或去除表转录组标记的细胞因子。总之，这项研究旨在确定 表转录组基因调控如何调节HIV-1复制，并开始揭示环境如何影响HIV-1复制， 包括滥用药物在内的各种因素会影响这一进程。

ABSTRACT While the epitranscriptomic regulation of viral RNA function has begun to attract considerable attention, this work has so far focused entirely on a single epitranscriptomic modification, i.e., the addition of a methyl group to the N6 position of adenosine (m6A). This focus is understandable, given that m6A is the most common epitranscriptomic modification of cellular mRNAs and it has been known for ~40 years that viral transcripts also contain high levels of m6A. Moreover, we and others have reported that m6A regulates HIV-1 replication and we have recently extended this work by showing that m6A also enhances the replication and pathogenicity of influenza A virus (IAV). A second epitranscriptomic modification, the isomerization of uridine to pseudouridine (ψ), previously thought to be confined to non-coding RNAs, has recently been shown to be almost as prevalent on cellular mRNAs as m6A and we have now observed that the genomic RNAs (gRNAs) packaged into virions of the retrovirus murine leukemia virus display a range of epitranscriptomic modifications, including not only m6A and ψ but also 5-methylcytosine (5mC) residues. In this grant application, we propose to use ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to systematically identify and quantitate the epitranscriptomic modifications that are present on highly purified gRNAs isolated from HIV-1 virions. We will then map the precise locations of three of the most prevalent epitranscriptomic modifications on both the gRNA and on HIV-1 mRNAs expressed in CD4+ T-cells and myeloid cells. Next, we will seek to silently mutate these modified sites in the context of an infectious HIV-1 provirus and determine whether, and how, these modifications affect viral gene expression and replication. We will also seek to identify the cellular factors that deposit these epitranscriptomic marks on HIV-1 transcripts and we will then test the effect of loss of expression, or overexpression, of these factors on HIV-1 replication in CD4+ T cells. Finally, we will analyze the effect of environmental stresses, such as heat shock and treatment with drugs of abuse, specifically nicotine and morphine, on the level of epitranscriptomic modification of cellular and viral transcripts expressed in HIV-1 infected cells. In parallel, we will also determine whether these same treatments affect the level of expression of the cellular factors that add or remove epitranscriptomic marks. Together, this research is designed to determine how epitranscriptomic gene regulation modulates HIV-1 replication and begin to shed light on how environmental factors, including drugs of abuse, can affect this process.