Project 2

项目2

• 批准号: 10506988
• 负责人: ALAN D FRANKEL
• 金额: $ 158.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10506988
• 关键词: AIDS/HIV problem; Address; Architecture; Binding; Biochemical; Biological Assay; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Carrier Proteins; Cell Line; Cell model; Cells; Chromatin; Chromatin Remodeling Factor; Chronic; Clonal Expansion; Clone Cells; Complex; Cryoelectron Microscopy; Development; Dimerization; Drug Targeting; Evolution; Gene Expression; Genetic; Genetic Transcription; Genetic Translation; HIV; HIV-1; HIV-2; Heterochromatin; Histones; Individual; Infection; Integration Host Factors; Introns; Link; Lymphocyte; Maintenance; Messenger RNA; Modeling; Modification; Molecular; Molecular Conformation; NF-kappa B; Nuclear Export; Nuclear Pore Complex Proteins; Pathway interactions; Peptides; Persons; Phase; Play; Positive Transcriptional Elongation Factor B; Post-Translational Protein Processing; Process; Property; Protein Isoforms; Proteins; Proteomics; Proviruses; RNA; RNA Binding; RNA Splicing; RNA Transport; Regulation; Reporter; Reporting; Research Priority; Resolution; Response Elements; Rest; Role; SIV; Shock; Signal Pathway; Signal Transduction; Site; Small Nuclear Ribonucleoproteins; Structure; TRAF6 gene; Testing; Time; Transcript; Transcription Elongation; Transcription Initiation; Ubiquitination; United States National Institutes of Health; Viral; Viral Genes; Virus; Virus Integration; Virus Replication; X-Ray Crystallography; biophysical techniques; dimer; experimental study; genetic analysis; genetic regulatory protein; genomic locus; inhibitor; integration site; latent HIV reservoir; latent infection; mRNA Export; macrophage; monocyte; mutant; mutation screening; novel; promoter; recruit; stem; structural biology; synergism; tat Protein; transcription factor; ubiquitin ligase; ubiquitin-protein ligase; viral RNA

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES PROJECT 2: REGULATION OF HIV TRANSCRIPTION AND LATENCY SUMMARY The HIV regulatory proteins Tat and Rev play a crucial role in controlling HIV transcription and mRNA export, respectively. In Project 2, we will functionally and structurally elucidate the roles of ubiquitin ligases that modify Tat, reveal the architecture of the Rev/RRE nuclear export complex to understand the mechanisms regulating HIV mRNA translation, and compare Rev and RRE evolution in SIV and HIV to identify viral factors that contribute to zoonosis. To achieve this, we will determine the functional interactions between the E3 ligases UBE2O and TRAF6 with Tat by cryo-EM or X-ray crystallography (Structural Biology Core) and investigate the mechanism by which UBE2O promotes the release of the inhibitory 7SK snRNP by ubiquitination of the HEXIM1 subunit. We will determine the high-resolution structure of the Rev/RRE/Crm1/RanGTP nuclear export complex by cryo- EM and define genetic and protein interaction landscapes of the Rev/RRE complex. We will also compare its protein-protein and protein-RNA interfaces between HIV-1, HIV-2, and SIV complexes. We will use the HEPS platform (Genetics core, Proteomics core and Structural biology core) and deep mutational scanning to functionally validate these interactions and define them biochemically and structurally. The latent HIV reservoir represents a significant roadblock to eradicating infection. We aim to uncover factors that drive HIV latency, including virus integration sites and the states of chromatin and chromatin interacting proteins. Using CRISPR-Cas9 (Genetics core), we will integrate a minimal HIV-1 LTR reporter into CD4+ T cells to address how integration into specific genomic loci may permit expansion of particular clonal cells without virus expression, to understand mechanistically how a large fraction of latent cells expand in chronically infected individuals over time. Using proteomics and a variety of biochemical and biophysical methods, we will identify post-translational modifications on HP1 proteins, test the roles of specific HP1 modifications on their phase- separation properties, and assess site-specific HP1 mutants for virus replication. This will elucidate how the state of chromatin and heterochromatin impact HIV transcription. Finally, we will examine the role of non-canonical (nc) NF-kB in enhancing latency reversal for shock-and-kill therapies. We will assess synergies between inhibition of SAMHD1 and of the ncNF-kB pathway in latency reversal in monocytic and lymphocytic latent cell models, resting CD4+ T cells (the major reservoir of latent HIV), and gut macrophages isolated from people living with HIV (PLWH). In summary, Project 2 will uncover potential new interfaces as HIV drug targets and evaluate latency reversal or induction of deep latency for cure strategies.

HARC 中心：HIV 配件和调节复合体 项目 2：HIV 转录和潜伏期的调控 概括 HIV 调节蛋白 Tat 和 Rev 在控制 HIV 转录和 mRNA 输出方面发挥着至关重要的作用， 分别。在项目2中，我们将从功能和结构上阐明修饰泛素连接酶的作用 Tat，揭示 Rev/RRE 核输出复合体的架构，以了解调节机制 HIV mRNA 翻译，并比较 SIV 和 HIV 中的 Rev 和 RRE 进化，以确定有助于识别的病毒因素 人畜共患病。为了实现这一目标，我们将确定 E3 连接酶 UBE2O 和 TRAF6 与 Tat 通过冷冻电镜或 X 射线晶体学（结构生物学核心）并研究其机制 UBE2O 通过 HEXIM1 亚基的泛素化促进抑制性 7SK snRNP 的释放。 我们将通过冷冻确定 Rev/RRE/Crm1/RanGTP 核输出复合物的高分辨率结构 EM 并定义 Rev/RRE 复合体的遗传和蛋白质相互作用景观。我们还将比较其 HIV-1、HIV-2 和 SIV 复合物之间的蛋白质-蛋白质和蛋白质-RNA 界面。我们将使用 HEPS 平台（遗传学核心、蛋白质组学核心和结构生物学核心）和深度突变扫描 从功能上验证这些相互作用并从生化和结构上定义它们。 潜在的艾滋病毒储存库是根除感染的一个重大障碍。我们的目标是揭示因素 驱动 HIV 潜伏期的因素，包括病毒整合位点以及染色质和染色质相互作用的状态 蛋白质。使用 CRISPR-Cas9（遗传学核心），我们将把最小的 HIV-1 LTR 报告基因整合到 CD4+ T 细胞中 解决整合到特定基因组位点如何允许在没有病毒的情况下扩增特定克隆细胞 表达，以从机制上了解大部分潜伏细胞如何在慢性感染中扩增 个人随着时间的推移。使用蛋白质组学和各种生物化学和生物物理方法，我们将识别 HP1 蛋白的翻译后修饰，测试特定 HP1 修饰对其相的作用 分离特性，并评估病毒复制的位点特异性 HP1 突变体。这将阐明国家如何 染色质和异染色质的影响 HIV 转录。最后，我们将研究非规范的作用 (nc) NF-kB 增强电击杀伤疗法的潜伏期逆转。我们将评估之间的协同效应 抑制 SAMHD1 和 ncNF-kB 通路在单核细胞和淋巴细胞潜伏细胞潜伏期逆转中的作用 模型、静息 CD4+ T 细胞（潜在 HIV 的主要储存库）以及从活人体内分离出的肠道巨噬细胞 艾滋病毒感染者（PLWH）。总之，项目 2 将发现作为 HIV 药物靶点的潜在新界面并评估 潜伏期逆转或诱导深度潜伏期以用于治疗策略。