Core 2

HARC中心：HIV附件和调控复合体 遗传学核心 摘要 为了支持所有HARC中心项目，Genetics Core将提供创新的高通量方法 原代细胞中HIV-宿主蛋白复合体的发现及功能鉴定 原代CD4+T细胞、静息原代CD4+T细胞和原代巨噬细胞)。与 蛋白质组学、结构生物学和计算核心，我们将开发HARC内源蛋白 用于不同HIV宿主蛋白发现和功能鉴定的结构(HEPS)平台 通过在基因组位置插入亲和标签以允许亲和纯化内源蛋白质复合体 直接从感染艾滋病毒的原代细胞中分离出来。使用我们突破性的CRISPR基因敲除(CRISPRko)，激活 (CRISPRa)、干扰(CRISPRi)和敲入(CRISPRki)技术，我们将(1) 生成前病毒和抗病毒HIV相互作用宿主因素的系统图，(2)功能验证新 HIV-宿主相互作用，(3)对特定蛋白结构域进行内源性深度突变扫描以识别 关键的宿主-病原体相互作用界面，以及(4)在目标位置引入/替换小片段DNA 基因组位置来研究特定遗传变异的功能。总的来说，这些基因研究将加强 HARC的中心任务是了解HIV辅助蛋白和宿主因子之间的相互作用，并开放 为更广泛的艾滋病毒研究界开辟新的实验和治疗途径。总而言之，遗传学 CORE将开发用于基因扰动的工具箱，以补充创新的结构生物学和蛋白质组学 努力。

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES GENETICS CORE SUMMARY To support all HARC Center Projects, the Genetics Core will provide innovative high-throughput methods for the discovery and functional characterization of HIV-host protein complexes in primary cells (i.e. activated primary CD4+ T cells, resting primary CD4+ T cells, and primary macrophages). Working in tandem with the Proteomics, Structural Biology, and Computational Cores, we will develop the HARC endogenous protein structure (HEPS) platform for the discovery and functional characterization of different HIV-host protein complexes by inserting affinity tags at genomic loci to allow affinity purification of endogenous protein complexes directly from HIV infected primary cells. Using our breakthrough CRISPR knock-out (CRISPRko), activation (CRISPRa), interference (CRISPRi), and knock-in (CRISPRki) technologies in primary human T cells, we will (1) generate a systematic map of proviral and antiviral HIV-interacting host factors, (2) functionally validate novel HIV-host interactions, (3) undertake endogenous deep mutational scanning of specific protein domains to identify critical host-pathogen interaction interfaces, and (4) introduce/replace small stretches of DNA at targeted genomic sites to study the function of specific genetic variants. Collectively, these genetic studies will strengthen the central HARC mission to understand interactions between HIV accessory proteins and host factors and open new experimental and therapeutic avenues for a broader HIV research community. In summary, the Genetics Core will develop a toolbox for genetic perturbations to complement innovative structural biology and proteomic efforts.