The Role of APOBEC3A in HIV Restriction

项目摘要/摘要 APOBEC3家族(APOBEC3A-H)由7个单链DNA胞苷脱氨酶组成 蛋白质(APOBEC3A-H)。这些蛋白质对包括艾滋病毒在内的各种病毒起到限制因子的作用。四 在这些蛋白质中，APOBEC3D-H已知通过包装成病毒粒子和突变的单链DNA来限制HIV 在下一轮感染的逆转录过程中。 APOBEC3A(A3A)因其独特的、高度的 限制性表达：仅在髓系细胞(巨噬细胞、单核细胞和树突状细胞)中表达 对干扰素刺激的反应。此外，与其他APOBEC3蛋白不同的是，A3A没有包装成 病毒粒子，并已被证明在干扰素刺激的原代单核细胞中阻断HIV复制的早期步骤 巨噬细胞指向一种新的、非规范的限制机制。APOBEC3A也不相互作用 Vif，一种HIV辅助蛋白，通过标记其他APOBEC3蛋白来对它们产生抵抗力 蛋白酶体的降解。目前对A3A如何靶向HIV及其表达方式知之甚少 以如此不同寻常的方式进行监管。 我们提出了两个特定的目标来阐明A3A针对HIV的机制以及它是如何 表达在分子水平上受到控制。在具体目标1中，我们将剖析A3A在艾滋病毒中所扮演的角色 感染。在初步研究中，我们使用了基于CRISPR/Cas9的协同激活中介系统 在人类T细胞中特异性地激活内源性A3A转录，而T细胞通常不表达A3A 细胞内抑制物。我们将使用这些经过改造的T细胞株来测试其作用程度和作用模式 A3A通过其靶向HIV感染。使用这个相同的系统，我们还将检查A3A表达式是否单独 足以使初级T细胞对艾滋病毒产生抵抗力。我们还将在原始人身上进行基因敲除实验 使用电穿孔介导的CRISPR核糖核蛋白(CrRNP)传递单核细胞。这允许 有效地敲除这些细胞中的基因。在具体目标2中，我们建议研究控制 A3A独特的表达模式。使用公开可用的大型数据集，我们确定了 A3A转录起始点是A3A所特有的，并且不在任何其他APOBEC3编码的上游 区域。我们将克隆这些感兴趣的区域，并使用不同的方法，包括荧光素酶分析来解剖 这些区域如何影响A3A转录。A3A基因调控的变异可能影响对HIV的易感性 同时也是多种人类恶性肿瘤的危险因素。 综上所述，我们的发现将塑造我们对行动模式、监管和 这一强大的细胞内DNA突变体蛋白在抗病毒免疫和免疫反应中的生物学功能 癌症。

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.