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: 8139592
• 负责人: QIANG ZHOU
• 金额: $ 37.68万
• 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 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139592
• 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; 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. PUBLIC HEALTH RELEVANCE: Latent reservoirs of HIV are the principal impediment to eradication of infection as they harbor silent proviruses that resume active replication once therapy is disrupted. This proposal explores the potential of targeting our recently identified human cofactors that are important for HIV transcription to reactivate latent viruses, which can then be cleared by highly active antiretroviral treatment (HAART). The proposed studies may enable the development of novel adjunctive therapeutic strategies to specifically and efficiently eradicate latent HIV reservoirs in infected patients.

描述(申请人提供)：HIV-1的潜伏期是根除感染的主要障碍，因为它们含有转录沉默的前病毒，一旦治疗中断，这些前病毒就会恢复复制。正在开发通过在HAART存在的情况下重新激活潜伏的艾滋病毒来清除这些宿主的方法。然而，现有潜伏期激活剂的有效性和特异性还需要大幅提高，这只能通过鉴定和表征其相关的分子靶标来实现(S)。这项建议探索了以我们最近发现的TAT辅助因子为目标来激活延迟的可能性。一种被广泛研究的TAT辅因子是P-TEFb，其活性形式最近被证明存在于一种名为BFEC(双功能延伸复合体)的新型复合体中，该复合体还包含ELL2、AFF4、ENL和AF9。在BFEC中，AFF4作为连接P-TEFb和ELL2的支架，这两个众所周知的转录延伸因子通过不同的机制发挥作用。这种协同作用激活了许多细胞和病毒启动子的延长，尽管最显著的影响是在HIV LTR上。重要的是，TAT与BFEC结合，显著促进BFEC的形成，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一种短命蛋白质。TAT/AFF4促进的BFEC的形成在需要ELL2‘S磷酸化的过程中稳定ELL2，可能是通过P-TEFb。最后，BFEC在HIV潜伏期激活中起关键作用，Prostratin，HMBA和SAHA，这三个研究最多的潜伏期化学激活剂，被发现类似于TAT促进ELL2的表达和与P-TEFb的相互作用。这些发现支持核心假设，即BFEC的功能和形成可以促进重新激活潜伏的HIV。为了测试这一点，我们将检验活性的BFEC是否既是从潜伏感染的T细胞系和原代CD4细胞重新激活HIV的必要条件和充分条件。为了产生高效抗降解的ELL2潜伏期激活，并控制上游信号通路以进一步增强这一作用，我们将确定ELL2的磷酸化位点(S)和控制ELL2稳定性和BFEC形成的负责激酶(S)。最后，为了阐明导致ELL2降解的蛋白降解途径，我们将测试可以被TAT诱导的磷酸化抑制的ELL2泛素化是否通过蛋白酶体触发ELL2降解。主要工作还将针对鉴定ELL2特有的泛素化酶，这可能揭示可以被抑制以稳定ELL2以有效形成BFEC的靶点。总之，拟议的研究可能使开发新的辅助治疗策略，以专门和有效地消除艾滋病毒患者的潜在宿主。 与公共卫生相关：艾滋病毒的潜伏宿主是根除感染的主要障碍，因为它们藏匿着沉默的前病毒，一旦治疗中断，这些前病毒就会恢复活跃的复制。这项建议探索了以我们最近发现的人类辅因子为靶点的可能性，这些辅因子对艾滋病毒转录具有重要作用，以重新激活潜伏病毒，然后通过高效抗逆转录病毒治疗(HAART)清除潜伏病毒。拟议的研究可能有助于开发新的辅助治疗策略，以特定和有效地消除感染患者中潜在的艾滋病毒宿主。