Tat cofactors and control of HIV-1 latency

Tat辅助因子和HIV-1潜伏期的控制

• 批准号: 8811092
• 负责人: QIANG ZHOU
• 金额: $ 37.36万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-07 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8811092
• 关键词: 26S proteasome; AIDS/HIV problem; Anti-Retroviral Agents; Binding; Biology; CD4 Positive T Lymphocytes; Cell Line; Chemicals; Chronic; Complex; Development; Elongation Factor; Enzymes; Generations; Genetic Transcription; HIV; HIV-1; Health; Hexamethylene Bisacetamide; Human; Immune system; Infection; Interruption; Latent Virus; Life; Life Cycle Stages; MLLT3 gene; Methods; Molecular; Molecular Target; Pathway interactions; Patients; Phosphorylation; Phosphorylation Site; Phosphotransferases; Polymerase; Positive Transcriptional Elongation Factor B; Process; Proteins; Proviruses; Resistance; Rest; Role; Signal Pathway; Specificity; T-Lymphocyte; Testing; Therapeutic; Transactivation; Transcription Elongation; Ubiquitination; Viral; Vorinostat; Work; base; cofactor; multicatalytic endopeptidase complex; novel; promoter; prostratin; purge; scaffold; upstream kinase

DESCRIPTION (provided by applicant): Latent reservoirs of HIV-1 are the principal impediment to eradication of infection as they harbor transcriptionally silent proviruses that resume replication once therapy is disrupted. Methods are being developed to purge these reservoirs through reactivating latent HIV in the presence of HAART. However, the efficacy and specificity of the available latency activators are in need of major improvement, which can only be achieved through the identification and characterization of their relevant molecular target(s). This proposal explores the potential of targeting our recently identified Tat cofactors to activate latency. One widely studied Tat cofactor is P-TEFb, whose active form was recently shown to exist in a novel complex termed BFEC (bi-functional elongation complex) that also contains ELL2, AFF4, ENL and AF9. Within BFEC, AFF4 works as a scaffold to interconnect P-TEFb and ELL2, two well-known transcription elongation factors that act by distinct mechanisms. This synergistically activates elongation from many cellular and viral promoters, although the most prominent effect is on the HIV LTR. Importantly, Tat binds to BFEC to markedly enhance its formation and coordinate the actions of P-TEFb and ELL2 on the same polymerase enzyme to stimulate HIV transcription. ELL2 is normally a short-lived protein targeted by the proteasome. The Tat/AFF4-promoted BFEC formation stabilizes ELL2 in a process that requires ELL2's phosphorylation by probably P-TEFb. Finally, implicating a key role for BFEC in HIV latency activation, prostratin, HMBA and SAHA, the three most highly studied chemical activators of latency, are found to act like Tat to promote ELL2 expression and interaction with P-TEFb. These findings support the central hypothesis that the function and formation of BFEC can be promoted to reactivate latent HIV. To test this, we will examine whether active BFEC is both necessary and sufficient to reactivate HIV from latently infected T cell lines and primary CD4 cells. To generate degradation-resistant ELL2 highly potent for latency activation and control the upstream signaling pathway to further enhance this effect, we will identify the phosphorylation site(s) in ELL2 and the responsible kinase(s) that controls ELL2 stability and BFEC formation. Finally, to elucidate the proteolytic pathway that causes ELL2 degradation, we will test whether the ubiquitination of ELL2, which can be suppressed by Tat-induced phosphorylation, triggers ELL2 degradation by the proteasome. Major efforts will also be directed toward the identification of the ubiquitination enzymes specific for ELL2, which may reveal targets that can be inhibited to stabilize ELL2 for efficient BFEC formation. Together, the proposed studies may enable the development of novel adjunctive therapeutic strategies to specifically and efficiently eradicate latent reservoirs in HIV patients.

描述（由申请人提供）：HIV-1的潜伏宿主是根除感染的主要障碍，因为它们携带转录沉默的前病毒，一旦治疗中断，它们就会恢复复制。正在开发通过在HAART治疗下重新激活潜伏的HIV来清除这些储存库的方法。然而，现有的潜伏期激活剂的疗效和特异性还需要很大的提高，这只能通过对其相关分子靶点的鉴定和表征来实现。本提案探讨了针对我们最近发现的Tat辅助因子激活潜伏期的潜力。一种广泛研究的Tat辅助因子是P-TEFb，其活性形式最近被证明存在于一种称为BFEC（双功能延伸复合物）的新型复合物中，该复合物还包含ELL2， AFF4， ENL和AF9。在BFEC中，AFF4作为支架连接P-TEFb和ELL2，这两种众所周知的转录延伸因子通过不同的机制起作用。这协同激活了许多细胞和病毒启动子的延伸，尽管最显著的作用是在HIV LTR上。重要的是，Tat与BFEC结合，显著增强其形成，并协调P-TEFb和ELL2在同一聚合酶上的作用，以刺激HIV转录。ELL2通常是蛋白酶体靶向的短寿命蛋白。Tat/ aff4促进的BFEC形成在一个可能需要ELL2被P-TEFb磷酸化的过程中稳定ELL2。最后，BFEC在HIV潜伏期激活中发挥关键作用，prostratin， HMBA和SAHA这三种研究最多的潜伏期化学激活剂被发现像Tat一样促进ELL2表达并与P-TEFb相互作用。这些发现支持了一个中心假设，即BFEC的功能和形成可以促进潜伏性HIV的重新激活。为了验证这一点，我们将检查活跃的BFEC是否对从潜伏感染的T细胞系和原代CD4细胞中重新激活HIV既是必要的，也是充分的。为了产生高活性的降解抗性ELL2，并控制上游信号通路以进一步增强这种作用，我们将确定ELL2中的磷酸化位点和控制ELL2稳定性和BFEC形成的激酶。最后，为了阐明导致ELL2降解的蛋白水解途径，我们将测试ELL2的泛素化（可被tat诱导的磷酸化抑制）是否触发了蛋白酶体对ELL2的降解。研究人员还将致力于鉴定ELL2特异性的泛素化酶，这可能会揭示出可以抑制ELL2以稳定ELL2以有效形成BFEC的靶标。总之，提出的研究可能有助于开发新的辅助治疗策略，以特异性和有效地根除HIV患者的潜伏库。