The Role of APOBEC3A in HIV Restriction

APOBEC3A 在 HIV 限制中的作用

• 批准号: 9974273
• 负责人: Emma Lynn McGregor
• 金额: $ 1.65万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2020-07-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9974273
• 关键词: Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Models; Biological Process; Biology; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Death; Cell Line; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Complement; Complementary DNA; Computers; Cytidine; Cytidine Deaminase; DNA; Data; Data Set; Dendritic Cells; Electroporation; Expression Profiling; Family; Family member; Fellowship; Generations; Genetic Transcription; Genome; Genomic Segment; HIV; HIV Infections; Human; IRF1 gene; Infection; Interferon Type I; Interferons; Knock-out; Laboratories; Life Cycle Stages; Luciferases; Malignant Neoplasms; Mediating; Mediator of activation protein; Mentors; Molecular; Molecular Virology; Mutate; Myeloid Cells; National Institute of Allergy and Infectious Disease; Open Reading Frames; Pathway interactions; Pattern; Play; Predisposition; Principal Investigator; Proteins; Proviruses; Publishing; Regulation; Reporting; Research Training; Resistance; Reverse Transcription; Ribonucleoproteins; Risk Factors; Role; Scientist; Shapes; Single-Stranded DNA; System; T-Lymphocyte; Testing; Transcription Initiation Site; United States National Institutes of Health; Up-Regulation; Uracil; Variant; Viral; Virion; Virus; Virus Replication; Work; antiviral immunity; base; engineered T cells; experimental study; genotoxicity; inhibitor/antagonist; insight; interest; knock-down; knockout gene; large datasets; macrophage; monocyte; multicatalytic endopeptidase complex; next generation; novel; overexpression; p65; response; selective expression; skills; stable cell line; transcription factor; treatment strategy; vif Gene Products

PROJECT SUMMARY/ABSTRACT The APOBEC3 family (APOBEC3A-H) of single-stranded DNA cytidine deaminases consists of seven proteins (APOBEC3A-H). These proteins act as restriction factors against a variety of viruses including HIV. Four of these proteins, APOBEC3D-H, are known to restrict HIV by being packaged into virions and mutating ssDNA during reverse transcription during the next round of infection. APOBEC3A (A3A) stands out against the other APOBEC3 family members because of its unique, highly restricted expression: it is expressed only in myeloid cells (macrophages, monocytes, and dendritic cells) in response to interferon stimulation. Moreover, unlike the other APOBEC3 proteins, A3A is not packaged into virions and has been shown to block the early steps of HIV replication in interferon stimulated primary monocytes and macrophages pointing to a novel, non-canonical mechanism of restriction. APOBEC3A also does not interact with Vif, an HIV accessory protein which confers resistance to other APOBEC3 proteins by marking them for degradation in the proteasome. Little is currently known about how A3A targets HIV and how its expression is regulated in such an unusual manner. We propose two Specific Aims to elucidate the mechanism by which A3A targets HIV and how its expression is controlled at the molecular level. In Specific Aim 1, we will dissect the role A3A plays in HIV infection. In preliminary studies, we have used the CRISPR/Cas9-based synergistic activation mediator system to specifically activate endogenous A3A transcription in human T cells, which generally fail to express this intracellular inhibitor. We will use these engineered T cell lines to test the degree to which and the mode of action by which A3A targets HIV infection. Using this same system, we will also examine if A3A expression alone is sufficient to render primary T cells resistant to HIV. We will also conduct knockout experiments in primary human monocytes using electroporation-mediated delivery of CRISPR ribonucleoprotein (crRNP). This allows for efficient knockout of genes in these cells. In Specific Aim 2, we propose to examine the factors controlling the unique expression pattern of A3A. Using publicly available large datasets, we identify regions upstream of the A3A transcription start site that are unique to A3A and are absent upstream of any other APOBEC3 coding region. We will clone these regions of interest and use different approaches including luciferase assays to dissect how these regions influence A3A transcription. Variation in regulation of A3A may influence susceptibility to HIV as well as represent a risk factor for a variety of human malignancies. Taken together, our findings will shape our understanding of the mode of action, the regulation and the biological function of this powerful intra-cellular DNA mutator protein at the interface of antiviral immunity and cancer.

项目总结/摘要 单链DNA胞苷脱氨酶的APOBEC 3家族（APOBEC 3A-H）由七个组成 蛋白（APOBEC 3A-H）。这些蛋白质作为限制因子对抗包括HIV在内的多种病毒。四 已知这些蛋白质中的APOBEC 3D-H通过包装成病毒体并使ssDNA突变来限制HIV 在下一轮感染的逆转录过程中。 APOBEC 3A（A3 A）与其他APOBEC 3家族成员相比脱颖而出，因为它具有独特的，高度的 限制性表达：仅在骨髓细胞（巨噬细胞、单核细胞和树突细胞）中表达， 对干扰素刺激的反应。此外，与其他APOBEC 3蛋白不同，A3 A不被包装到 已显示阻断干扰素刺激的原代单核细胞中HIV复制的早期步骤 和巨噬细胞指出了一种新的，非经典的限制机制。APOBEC 3A也不相互作用 Vif是一种HIV辅助蛋白，它通过标记其他APOBEC 3蛋白， 蛋白酶体的降解。目前对A3 A如何靶向HIV以及其表达如何影响HIV的研究还知之甚少。 以如此不寻常的方式进行管理。 我们提出了两个具体的目的，以阐明A3 A靶向HIV的机制，以及它如何与HIV结合。 在分子水平上控制表达。在具体目标1中，我们将剖析A3 A在艾滋病毒中的作用 感染在初步研究中，我们使用了基于CRISPR/Cas9的协同激活介体系统， 特异性激活人T细胞中的内源性A3 A转录，而人T细胞通常不能表达这种A3 A转录。 胞内抑制剂我们将使用这些工程化的T细胞系来测试其作用程度和作用模式。 A3 A通过其针对艾滋病毒感染。使用同一系统，我们还将检查A3 A表达式是否单独 足以使原代T细胞抵抗HIV。我们还将在原代人类中进行基因敲除实验， 使用电穿孔介导的CRISPR核糖核蛋白（crRNP）递送，在单核细胞中表达CRISPR。这允许 有效地敲除这些细胞中的基因。在具体目标2中，我们建议检查控制 A3 A的独特表达模式。使用公开可用的大型数据集，我们确定了 A3 A转录起始位点是A3 A独有的，并且在任何其他APOBEC 3编码上游不存在 地区我们将克隆这些感兴趣的区域，并使用不同的方法，包括荧光素酶测定， 这些区域如何影响A3 A转录。A3 A调节的变化可能影响对HIV的易感性 也是多种人类恶性肿瘤的危险因素。 总之，我们的研究结果将塑造我们的理解的行动模式，监管和 这种强大的细胞内DNA突变蛋白在抗病毒免疫界面的生物学功能， 癌