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：艾滋病毒附件和调节复合物 项目2：调节艾滋病毒的传播和潜伏期 总结 HIV调节蛋白达特和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泛素连接酶的作用， 达特，揭示Rev/RRE核输出复合体的结构，以了解调节 HIV mRNA翻译，并比较SIV和HIV中Rev和RRE的进化，以确定有助于 人畜共患病为了实现这一点，我们将确定E3连接酶UBE 2 O和 TRAF 6与达特通过冷冻EM或X射线晶体学（结构生物学核心），并研究其机制 UBE 2 O通过泛素化HEXIM 1亚基促进抑制性7SK snRNP的释放。 我们将通过低温冷冻技术确定Rev/RRE/Crm 1/RanGTP核出口复合物的高分辨率结构， EM和定义Rev/RRE复合物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，并从生物化学和结构上定义它们。 潜伏的艾滋病毒储存库是消除感染的一个重大障碍。我们的目标是揭示 驱动HIV潜伏期，包括病毒整合位点和染色质与染色质相互作用的状态 proteins.使用CRISPR-Cas9（遗传学核心），我们将最小的HIV-1 LTR报告基因整合到CD 4 + T细胞中， 为了解决如何整合到特定的基因组位点可以允许扩增特定的克隆细胞而不需要病毒， 表达，以了解大部分潜伏细胞如何在慢性感染中机械地扩增， 个人随着时间的推移。利用蛋白质组学和各种生物化学和生物物理方法，我们将确定 HP 1蛋白的翻译后修饰，测试特定HP 1修饰在其相位上的作用， 分离特性，并评估用于病毒复制的位点特异性HP 1突变体。这将阐明国家如何 染色质和异染色质对HIV转录的影响。最后，我们将研究非规范的作用， (nc)NF-kB在增强休克和杀死疗法的潜伏期逆转中的作用我们将评估 单核细胞和淋巴细胞潜伏细胞中SAMHD 1和ncNF-kB通路在潜伏期逆转中的抑制 模型，静息的CD 4 + T细胞（潜伏HIV的主要储存库），以及从生活的人中分离的肠道巨噬细胞。 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潜伏期用于治疗策略。