Project 2

项目2

• 批准号: 10666673
• 负责人: ALAN D FRANKEL
• 金额: $ 152.92万
• 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/10666673
• 关键词: Address; Architecture; Binding; Biochemical; Biological Assay; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Carrier Proteins; Cell Line; Cell Separation; Cell model; Cells; Chromatin; Chromatin Remodeling Factor; Chronic; Clonal Expansion; Complex; Cryoelectron Microscopy; Development; Dimerization; Drug Targeting; Evolution; Gene Expression; Genetic; Genetic Transcription; HIV; HIV-1; HIV-2; HIV/AIDS; Heterochromatin; Histones; Individual; Infection; Integration Host Factors; Introns; Link; Lymphocyte; Macrophage; Maintenance; Messenger RNA; Modeling; Modification; Molecular; Molecular Conformation; NF-kappa B; NFKB Signaling Pathway; Nuclear Export; Nuclear Pore Complex Proteins; Pathway interactions; Peptides; Phase; Play; Positive Transcriptional Elongation Factor B; Post-Translational Protein Processing; Process; Property; Protein Isoforms; Proteins; Proteomics; Provirus Integration; RNA; RNA Binding; RNA Splicing; RNA Transport; Regulation; Reporter; Reporting; Research Priority; Resolution; Response Elements; Rest; Role; Running; SIV; Shock; 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; lonely individuals; mRNA Export; mRNA Translation; 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配件和监管综合体 项目2：艾滋病毒转录和潜伏期的调节 概括 HIV调节蛋白TAT和REV在控制HIV转录和mRNA输出方面起着至关重要的作用， 分别。在项目2中，我们将在功能和结构上阐明修改的泛素连接酶的作用 TAT，揭示Rev/RRE核出口复合物的结构，以了解调节的机制 HIV mRNA翻译，并比较SIV和HIV中的REV和RRE演变，以鉴定有助于的病毒因素 对人畜共患病。为此，我们将确定E3连接酶Ube2O和 TRAF6与Cryo-EM或X射线晶体学（结构生物学核心）一起使用TRAF6，并研究了机制 ube2O通过甲基氢化亚基的泛素化促进了抑制性7SK SNRNP的释放。 我们将确定Cryo-的Rev/RRE/RRE/CRM1/RANGTP核出口复合物的高分辨率结构 EM并定义了Rev/RRE复合物的遗传和蛋白质相互作用景观。我们还将比较它的 HIV-1，HIV-2和SIV复合物之间的蛋白质蛋白质和蛋白-RNA界面。我们将使用HEP 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 在功能上验证这些相互作用并在生化和结构上定义它们。 潜在的HIV水库代表了根除感染的重大障碍。我们的目标是发现因素 驱动HIV潜伏期，包括病毒整合位点以及染色质和染色质的状态 蛋白质。使用CRISPR-CAS9（遗传学核心），我们将将最小HIV-1 LTR报告基因整合到CD4+ T细胞中 为了解决特定基因组基因局的集成如何允许在没有病毒的情况下扩展特定的克隆细胞 表达，从机械上理解大量的潜在细胞如何在慢性感染中扩展 随着时间的流逝。使用蛋白质组学以及多种生化和生物物理方法，我们将确定 对HP1蛋白的翻译后修饰，测试特定HP1修饰在其相位的作用 分离特性，并评估特异性的HP1突变体进行病毒复制。这将阐明状态 染色质和异染色质撞击HIV转录。最后，我们将研究非规范的作用 （NC）NF-KB在增强冲击和杀伤疗法的潜伏期逆转方面。我们将评估之间的协同作用 单核细胞和淋巴细胞潜伏细胞中潜伏期逆转的SAMHD1和NCNF-KB途径的抑制作用 模型，静止的CD4+ T细胞（潜在艾滋病毒的主要储藏室）和从生活的人中分离出来的肠道巨噬细胞 与艾滋病毒（PLWH）。总而言之，项目2将发现潜在的新界面作为HIV药物目标并评估 治愈策略的潜伏期逆转或诱导深层延迟。