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附件和调控复合体 项目2：艾滋病毒转录和潜伏期的调控 摘要 HIV调节蛋白TAT和REV在控制HIV转录和信使核糖核酸输出方面起着关键作用， 分别进行了分析。在项目2中，我们将从功能和结构上阐明泛素连接酶在修饰 TAT，揭示REV/RRE核出口复合体的架构，以了解调节机制 HIV mRNA的翻译，并比较SIV和HIV中REV和RRE的进化，以确定参与其中的病毒因素 人畜共患病。为了实现这一点，我们将确定E3连接酶UBE2O和UBE2O之间的功能相互作用 TRAF6与TAT的低温电子显微镜或X射线结晶学(结构生物学核心)，并探讨其机制 其中UBE2O通过HEXIM1亚基的泛素化促进抑制的7SK SnRNP的释放。 我们将用冷冻法确定REV/RRE/CRM1/RanGTP核出口复合体的高分辨结构。 EM和定义REV/RRE复合体的遗传和蛋白质相互作用图景。我们也会比较它的 HIV-1、HIV-2和SIV复合体之间的蛋白质-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。 潜伏的艾滋病毒携带者是根除感染的重要障碍。我们的目标是揭示各种因素 导致HIV潜伏期的因素，包括病毒整合部位以及染色质和染色质相互作用的状态 蛋白质。使用CRISPR-Cas9(Genetics Core)，我们将把一个最小的HIV-1 LTR报告整合到CD4+T细胞中 为了解决如何整合到特定的基因组位置可能允许特定的克隆细胞在没有病毒的情况下扩张 表达，以机械地了解在慢性感染中大部分潜伏细胞是如何扩张的 随着时间的推移，个体。利用蛋白质组学和各种生化和生物物理方法，我们将识别 HP1蛋白的翻译后修饰，测试特定的HP1修饰对其时相的作用- 分离特性，并评估针对病毒复制的特定部位HP1突变体。这将阐明国家是如何 染色质和异染色质影响HIV转录。最后，我们将研究非规范的角色 (NC)核因子-kB在提高休克和杀伤性疗法的潜伏期逆转中的作用。我们将评估两国之间的协同效应 抑制SAMHD1和ncNF-kB通路逆转单核细胞和淋巴细胞潜伏期 模型，静息的CD4+T细胞(潜伏HIV的主要储存库)，以及从活人身上分离的肠道巨噬细胞 艾滋病毒携带者(PLWH)。总而言之，项目2将发现潜在的新接口作为艾滋病毒药物靶标并评估 治疗策略的潜伏期逆转或诱导深度潜伏期。