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

摘要 虽然病毒RNA功能的表位转录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位转录修饰上，即添加一个甲基 组至腺苷(M6A)的N6位。这种关注是可以理解的，因为m6a是最常见的 细胞mRNA的表位转录修饰，人们已经知道大约40年来，病毒的转录产物也 含有高水平的m6A。此外，我们和其他人已经报道了m6A调节HIV-1复制，我们 最近扩展了这项工作，表明m6A还增强了病毒的复制和致病性 甲型流感病毒(IAV)。第二个表位转录修饰，尿苷异构化为假尿苷 以前被认为仅限于非编码RNA的(ψ)，最近被证明几乎同样普遍 我们现在已经观察到包装成病毒粒子的基因组RNA(GRNAs) 逆转录病毒的小鼠白血病病毒表现出一系列的表位转录修饰，不仅包括m6A 和ψ，但也有5-甲基胞嘧啶(5mC)残基。在这项拨款申请中，我们建议使用超高 高效液相色谱串联质谱(UPLC-MS/MS)系统鉴定和鉴定 对从HIV-1分离的高纯度gRNA的表位转录修饰进行量化 病毒粒子。然后我们将绘制出三个最流行的表位转录修饰的准确位置 GRNA和HIV-1mRNAs均在CD4+T细胞和髓系细胞中表达。接下来，我们将寻求默默地 在传染性HIV-1前病毒的背景下突变这些修饰的位点，并确定是否以及如何 修饰会影响病毒基因的表达和复制。我们还将寻求确定细胞因素， 将这些表位转录标记存放在HIV-1转录本上，然后我们将测试表达缺失的效果， 或过度表达，这些因素对HIV-1在CD4+T细胞中的复制。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细胞和病毒转录本表位转录修饰的影响 被感染的细胞。同时，我们还将确定这些相同的处理是否会影响 增加或移除表位转录标记的细胞因子。总而言之，这项研究旨在确定 表位转录基因调控如何调节HIV-1复制，并开始揭示环境 包括滥用药物在内的各种因素都会影响这一过程。

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.