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

HIV-1 转录本的表观转录组修饰：滥用药物的影响

基本信息

批准号：
10371249
负责人：
BRYAN R. CULLEN
金额：
$ 32.2万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-15 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10371249
关键词：
Adenosine Affect Applications Grants Attention CD4 Positive T Lymphocytes Cells Deposition Electroconvulsive Therapy Environmental Risk Factor Exposure to Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Goals Growth and Development function HIV Genome HIV-1 Heat-Shock Response High Pressure Liquid Chromatography Immune response Individual Infection Influenza A virus Knock-out Light Location Maps Measures Messenger RNA Modification Morphine Murine leukemia virus Mutagenesis Mutate Myeloid Cells Nicotine Nuclear Nucleotides Pathogenicity Pattern Pharmaceutical Preparations Phenotype Poly(A)+ RNA Positioning Attribute Post-Transcriptional Regulation Process Proteins Proviruses Pseudouridine Publishing RNA RNA Interference RNA Stability Regulation Reporting Research Role Silent Mutation Site Stress Techniques Testing Transcript Untranslated RNA Uridine Viral Viral Genes Viral Pathogenesis Virion Virus Virus Replication Work base cell growth design drug of abuse environmental stressor epitranscriptomics genomic RNA in vivo interest mRNA Export mRNA Expression methyl group mutant overexpression pressure tandem mass spectrometry viral RNA viral genomics

项目摘要

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.

抽象的虽然病毒RNA功能的表参数调节已开始引起人们的注意，但到目前小组到腺苷的N6位置（M6A）。鉴于M6A是最常见的，这种重点是可以理解的细胞mRNA的表面转录体修饰，并且已经知道了40年的病毒转录本。包含高水平的M6A。此外，我们和其他人报告M6A调节HIV-1复制，我们最近通过表明M6A还增强了这项工作的扩展流感病毒（IAV）。第二次表演组修饰，尿苷与假氨酸的异构化（ψ）以前被认为仅限于非编码RNA，最近已被证明几乎是普遍的在细胞mRNA上为M6a，我们现在观察到基因组RNA（GRNA）包装到病毒中逆转录病毒鼠白血病病毒显示出一系列的表面转录体修饰，不仅包括M6A 和ψ，但还保留了5-甲基胞嘧啶（5MC）。在此赠款申请中，我们建议使用超高性能液相色谱串联质谱法（UPLC-MS/MS）系统识别和定量从HIV-1分离出的高纯化的GRNA上呈现的表面参考的修饰病毒体。然后，我们将绘制三个最普遍的表面分类组的精确位置在CD4+ T细胞和髓样细胞中表达的GRNA和HIV-1 mRNA。接下来，我们将寻求默默在感染性的HIV-1病毒中突变这些修改的位点，并确定是否以及如何以及如何修饰会影响病毒基因表达和复制。我们还将寻求确定细胞因素在HIV-1转录本上沉积这些表转录组标记，然后我们将测试表达丧失的影响，或过表达，这些因素对CD4+ T细胞中HIV-1复制的因素。最后，我们将分析环境压力，例如热休克和用滥用药物的治疗，特别是尼古丁和吗啡，在HIV-1中表达的细胞和病毒转录本的表面转录体修饰水平感染细胞。同时，我们还将确定这些相同的处理是否影响添加或去除同意标记的细胞因子。这项研究旨在确定表面参考基因调控如何调节HIV-1复制并开始阐明环境包括滥用药物在内的因素会影响这一过程。