Roles of N-TEF and P-TEBb in HIV Latency

N-TEF 和 P-TEBb 在 HIV 潜伏期中的作用

基本信息

批准号：
7899486
负责人：
Boris Matija PETERLIN
金额：
$ 40.16万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-15 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7899486
关键词：
Acquired Immunodeficiency Syndrome Address Adverse effects Antibodies Antisense Oligonucleotides C-terminal Cell Cycle Cell Cycle Stage Cell Line Cells Chimera organism Chimeric Proteins Complex Cultured Cells Data Diabetes Mellitus Dominant-Negative Mutation Drug or chemical Tissue Distribution Elongation Factor Future Genetic Transcription Genome Growth HIV Highly Active Antiretroviral Therapy Human Immune response Immune system Individual Infection Interphase Cell Jurkat Cells Lead Lectin Length Lentivirus Vector Long Terminal Repeats Lymphoid Lymphokines Macaca mulatta Modeling Molecular Monkeys Mus Organism Patients Peripheral Blood Mononuclear Cell Phenotype Phorbol Esters Phosphorylation Play Post-Translational Protein Processing Primates Production Proteins Proviruses RNA RNA Interference RNA Polymerase II Recruitment Activity Reporting Rest Reverse Transcriptase Polymerase Chain Reaction Role Running SIV Serum Symbiosis System Therapeutic Intervention Time Tissues Transactivation Transcript Transcription Elongation Transgenic Mice Viral Viral Genome Viremia Virion Virus Work adeno-associated viral vector chromatin immunoprecipitation cytokine extracellular factor EF-P gene therapy macrophage monocyte mutant negative elongation factor novel therapeutics promoter response stem tissue culture tissue/cell culture verdin photosensitizer viral RNA virology virus core

项目摘要

The persistence of HIV in infected individuals on HAART remains an insurmountable obstacle to the treatment and cure of AIDS. HIV establishes reservoirs in many cells and tissues. Integrated proviruses are not transcribed and infected cells are not recognized by the immune system. Nevertheless, upon cellular activation, growth and proliferation, these latent proviruses can direct the synthesis of fully infectious virions that infect other cells. Thus, the uneasy symbiosis between HIV and the host persists. In this study, the hypothesis is that much of this proviral latency is maintained at the levels of elongation of viral transcription. Studies of infected individuals at seroconversion and of patients on HAART, revealed that a substantial proportion of HIV in circulating PBMC is transcribing only short transcripts that contain the transactivation response (TAR) RNA stem loop. Thus, transcription has initiated but not elongated. Upon cellular activation and/or the addition of Tat, which can enter cells freely, full-length viral transcripts accumulate and HIV replication is observed. Factors that contribute to this lack of transcriptional elongation are called negative transcription elongation factor (N-TEF). They are opposed by the positive transcription elongation factor b (P-TEFb) that phosphorylates the C-terminal domain of RNA polymerase II and subunits of N-TEF to modify the transcription complex for productive elongation. Tat and NFkB are central players that recruit P-TEFb to the HIVLTR. In this study, the contributions of N-TEF and P-TEFb to proviral latency will be dissected in great detail, using approaches with dominant negative proteins and RNAi. Moreover, secreted Tat and P-TEFb.Tat fusion proteins will be used to try to abrogate this transcriptional arrest on the HIVLTR. Although these studies will be carried out first in cell lines and PBMC from the rhesus macaque, future therapeutic intervention using gene therapy with Tat and P-TEFb.Tat chimeras are contemplated. During these studies, our human model of proviral latency will find resonance with SIV in monkeys. Similar studies to those that had been carried out in infected humans will be carried out in rhesus macaques, i.e. we shall look for short and long transcripts from the SIVLTR, examine effects of blocking N-TEF or supplying P-TEFb to monkey PBMC, and finally, develop strategies to secrete abundant Tat and P-TEFb.Tat chimeras in monkeys before infection with SIV and later, after seroconversion and optimal treatment of the rhesus macaques. These studies should reveal once and for all what role transcriptional elongation plays in the latency of HIV, what effects, if any Tat has on the organism, and if manipulating P-TEFb could play a role in eliminating the reservoir of HIV from the host.

接受HAART治疗的感染者体内HIV病毒的持续存在仍然是治疗和治疗过程中难以克服的障碍。治愈艾滋病。 HIV 在许多细胞和组织中建立储存库。整合的原病毒不被转录并且受感染的细胞不被免疫系统识别。然而，在细胞激活、生长和通过增殖，这些潜伏的原病毒可以指导合成完全感染性的病毒颗粒，从而感染其他细胞。因此，艾滋病毒和宿主之间不稳定的共生关系持续存在。在这项研究中，假设大部分前病毒潜伏期维持在病毒转录延伸的水平。对感染者的研究血清转化和 HAART 患者的研究表明，循环 PBMC 中的 HIV 很大一部分是仅转录包含反式激活反应 (TAR) RNA 茎环的短转录本。因此，转录已开始但未延长。一旦细胞激活和/或添加 Tat，它可以进入细胞自由活动，全长病毒转录物积累，并观察到 HIV 复制。造成这种情况的因素缺乏转录延伸的因子称为负转录延伸因子（N-TEF）。他们反对的是磷酸化 RNA C 末端结构域的正转录延伸因子 b (P-TEFb) 聚合酶 II 和 N-TEF 亚基修饰转录复合物以实现有效延伸。 Tat 和 NFkB 是招募 P-TEFb 加入 HIVLTR 的核心参与者。在本研究中，N-TEF 和 P-TEFb 对将使用显性失活蛋白和 RNAi 方法对原病毒潜伏期进行详细剖析。此外，分泌的 Tat 和 P-TEFb.Tat 融合蛋白将用于尝试消除这种转录停滞 HIVLTR。尽管这些研究将首先在恒河猴的细胞系和 PBMC 中进行，未来的治疗干预使用Tat和P-TEFb的基因治疗。Tat嵌合体被考虑。期间在这些研究中，我们的人类前病毒潜伏期模型将与猴子中的 SIV 产生共鸣。类似的研究在受感染的人类中进行的那些研究将在恒河猴中进行，即我们将寻找来自 SIVLTR 的短和长转录本，检查阻断 N-TEF 或向猴子提供 P-TEFb 的效果 PBMC，最后，制定了在猴子体内分泌丰富的 Tat 和 P-TEFb.Tat 嵌合体的策略 SIV 感染以及随后的恒河猴血清转化和最佳治疗后。这些研究应该一劳永逸地揭示转录延伸在 HIV 潜伏期中起什么作用，有什么影响（如果有的话） Tat 对生物体有影响，如果操纵 P-TEFb 可以在消除体内 HIV 储存库方面发挥作用，主持人。