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

N-TEF 和 P-TEBb 在 HIV 潜伏期中的作用

• 批准号: 6844100
• 负责人: Boris Matija PETERLIN
• 金额: $ 22.05万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844100
• 关键词: HIV infections; Macaca mulatta; RNA interference; cell line; chimeric proteins; genetic transcription; human tissue; latent virus infection; provirus; simian immunodeficiency virus; transcription factor

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 NF-kB 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艾滋病治疗和治愈的一个不可克服的障碍。 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、生长和增殖后，这些潜伏的原病毒可以指导感染其他细胞的完全感染性病毒粒子的合成。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。对血清转换的感染者和接受 HAART 的患者的研究表明，循环 PBMC 中很大一部分 HIV 只转录包含反式激活反应 (TAR) RNA 茎环的短转录物。因此，转录已经开始但并未延长。细胞激活和/或添加可以自由进入细胞的 Tat 后，全长病毒转录物就会积累，并观察到 ​​HIV 复制。导致转录延伸缺乏的因素称为负转录延伸因子（N-TEF）。他们反对的是 正转录延伸因子 b (P-TEFb)，磷酸化 RNA 聚合酶 II 的 C 末端结构域和 N-TEF 亚基，以修饰转录复合物以实现有效延伸。 Tat 和 NF-kB 是将 P-TEFb 招募到 HIVLTR 的核心参与者。在本研究中，将使用显性失活蛋白和 RNAi 方法详细剖析 N-TEF 和 P-TEFb 对原病毒潜伏期的贡献。此外，分泌的 Tat 和 P-TEFb.Tat 融合蛋白将用于尝试消除 HIVLTR 上的这种转录停滞。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。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。与在感染人类中进行的研究类似的研究将在恒河猴中进行，即我们将从 SIVLTR 中寻找短转录本和长转录本，检查阻断 N-TEF 或向猴 PBMC 提供 P-TEFb 的效果，以及最后，制定策略以在猴子中分泌丰富的 Tat 和 P-TEFb.Tat 嵌合体 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，Tat 对生物体有什么影响（如果有的话），以及操纵 P-TEFb 是否可以在消除宿主体内的 HIV 储存方面发挥作用。