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.

HAART受感染者的艾滋病毒的持久性仍然是治疗和治愈艾滋病的障碍。 HIV在许多细胞和组织中建立了储层。综合的病毒未转录，被感染的细胞未被免疫系统识别。然而，在细胞激活，生长和增殖后，这些潜在的预科病毒可以指导染色的完全感染其他细胞的完全感染性病毒。因此，艾滋病毒和宿主之间的不安共生。在这项研究中，该假设是大部分预示性 潜伏期保持在病毒转录的伸长水平。在血清转化和HAART上的感染个体的研究表明，循环PBMC中的HIV很大一部分仅转录包含反式激活反应（TAR）RNA茎环的短转录本。因此，转录启动但未伸长。当细胞激活和/或添加TAT时，可以自由进入细胞，观察到全长病毒转录本，并观察到HIV复制。导致缺乏转录伸长的因素称为负转录伸长因子（N-TEF）。他们反对 阳性转录因子B（P-TEFB）磷酸化RNA聚合酶II的C末端结构域和N-TEF的亚基，以修改转录复合物以进行生产伸长。 TAT和NF-KB是将P-TEFB招募到HIVLTR的中心参与者。在这项研究中，使用具有主要负蛋白和RNAi的方法，将详细阐述N-TEF和P-TEFB对病毒潜伏期的贡献。此外，分泌的TAT和P-TEFB.TAT融合蛋白将用于尝试废除HIVLTR上的转录停滞。尽管这些研究将首先在细胞系和恒河猴的PBMC中进行，但 考虑使用基因治疗与TAT和P-TEFB的未来治疗干预。在这些研究中，我们的人类前病潜伏期模型将在猴子中与SIV产生共鸣。与在受感染人类中进行的类似研究将在恒河猕猴中进行，即，我们将寻找SIVLTR的短期和长转录，检查阻断N-TEF或向Monkey PBMC提供P-TEFB的效果，最后，开发出秘密的tat tat和P-tefb.ttat Chimskeys tatkeys tatkeys tatkeys， 在血清转化和最佳治疗恒河猕猴之后，用SIV感染。这些研究应揭示一旦揭示转录伸长在艾滋病毒潜伏期中的作用，如果有任何tat对生物体有什么影响，以及是否对p-TEFB操纵P-TEFB是否可以在消除宿主中消除HIV的储层中发挥作用。