Control of P-TEFb biogenesis and HIV transcription in primary T-cells

原代 T 细胞中 P-TEFb 生物发生和 HIV 转录的控制

• 批准号: 10403547
• 负责人: JONATHAN KARN
• 金额: $ 40.25万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10403547
• 关键词: Address; Affinity; Antibodies; Behavior; Binding Sites; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Blood Circulation; CDK9 Protein Kinase; Cell Line; Cell model; Cells; ChIP-seq; Complex; DNA; Data Set; Development; Dissociation; Enzymes; Equilibrium; FRAP1 gene; Genes; Genetic Transcription; HIV; HIV Infections; Image; Immunofluorescence Immunologic; Immunoprecipitation; In Situ; In Vitro; Kinetics; Ligation; Location; Lymphoid Tissue; Mediating; Methods; Modeling; Modification; Molecular; Monitor; Mutation; Pathway interactions; Patients; Pharmacology; Positive Transcriptional Elongation Factor B; Post-Translational Protein Processing; Properdin; Protein Kinase C; Proteins; Proteomics; Proviruses; RNA; Receptor Signaling; Regulation; Reproducibility; Rest; Role; Signal Pathway; Signal Transduction; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TCR Activation; Time; Tissue Sample; Tissues; Transactivation; Transcription Elongation; Transcriptional Elongation Factors; Transcriptional Regulation; Translations; Viral; Work; antiretroviral therapy; base; cyclin T1; epigenetic silencing; experimental study; factor EF-P; fluorescence imaging; in vivo; inhibitor; memory CD4 T lymphocyte; peripheral blood; phenotypic biomarker; promoter; protein complex; response; snRNP Biogenesis; spatiotemporal; stem; tat Protein; transcriptome sequencing; virtual

Our understanding of HIV latency and persistence has been complicated by the small numbers of latently infected cells found in the circulation, the difficulty of obtaining comprehensive sets of tissue samples from patients, the lack of known phenotypic markers that can distinguish latently infected cells from uninfected ones, and limited information about the behavior of tissue reservoirs in vivo. Mechanistic studies, conducted primarily using cell line models of HIV latency, have shown that viral reactivation requires transactivation of epigenetically silenced proviruses by the viral Tat protein in complex with the host transcription elongation co-factors P-TEFb and the super elongation complex (SEC). Crucially for the study of HIV latency, additional P-TEFb control mechanisms exist in resting memory CD4+ T cells, where CycT1 protein levels are drastically reduced. We have also recently shown in primary T cells that CDK9 is present in an inactive state bound to Hsp90/Cdc37. Therefore, specific T-cell signaling pathways need to be activated in order to assemble a functional 7SK snRNP complex in primary cells. Using a refined highly reproducible primary cell model of HIV latency (the QUECEL model), we will address two key unsolved, but fundamental, questions on the transcriptional control of HIV latency: (1) How do T-cell signaling pathways regulate the assembly of P-TEFb, 7SK snRNP and the SEC in memory CD4+ T cells? (2) What Tat-dependent and independent T-cell molecular mechanisms allow for the exchange of P-TEFb from 7SK snRNP to the SEC and eventually to the latent HIV provirus? Our specific aims will investigate the regulation of the biogenesis and disassembly of 7SK snRNP by post- translational modifications and T-cell signaling pathways (Aim 1), apply fluorescence imaging of the spatiotemporal distribution and delivery of P-TEFb to the latent provirus (Aim 2) and define the biochemistry of the exchange of P-TEFb from 7SK snRNP during proviral reactivation (Aim 3). The key technological breakthrough, which distinguishes this work from virtually all previous studies of HIV transcription regulation is that we now have available reliable primary cell models for HIV latency and reactivation. Working with primary cells can be challenging since relatively limited numbers of cells are available. We therefore emphasize the use of imaging experiments and highly sensitive ChIP-Seq and RNA-Seq assays in the majority of our experiments. Defining the molecular and cell biological mechanisms leading to P-TEFb biogenesis and its transfer to the HIV promoter should provide the definitive identification of the pharmacological targets that is needed for the development of new and efficient classes of latency reversing agents.

我们对艾滋病毒潜伏期和持久性的理解因人数少而变得复杂