Tat cofactors and control of HIV-1 latency

描述（由申请人提供）：HIV-1的潜在储库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，它们就会恢复复制。正在开发通过在HAART存在下重新激活潜伏的HIV来清除这些储库的方法。然而，可用的潜伏期激活剂的功效和特异性需要重大改进，这只能通过鉴定和表征其相关分子靶标来实现。该提案探讨了针对我们最近确定的达特辅因子激活潜伏期的潜力。一种广泛研究的达特辅因子是P-TEFb，其活性形式最近被证明存在于称为BFEC（双功能延伸复合物）的新型复合物中，该复合物还含有ELL 2、AFF 4、ENL和AF 9。在BFEC中，AFF 4作为支架连接P-TEFb和ELL 2，这两种众所周知的转录延伸因子通过不同的机制起作用。这协同激活许多细胞和病毒启动子的延伸，尽管最突出的作用是对HIV LTR的影响。重要的是，达特与BFEC结合以显著增强其形成并协调P-TEFb和ELL 2对相同聚合酶的作用以刺激HIV转录。ELL 2通常是蛋白酶体靶向的短寿命蛋白。达特/AFF 4促进的BFEC形成在需要ELL 2可能通过P-TEFb磷酸化的过程中稳定ELL 2。最后，暗示BFEC在HIV潜伏期激活中的关键作用，发现三种最高度研究的潜伏期化学激活剂prostratin、HMBA和SAHA与达特一样起作用以促进ELL 2表达和与P-TEFb的相互作用。这些发现支持了核心假设，即BFEC的功能和形成可以被促进以重新激活潜伏的HIV。为了验证这一点，我们将研究是否有活性的BFEC是必要的和足够的重新激活潜伏感染的T细胞系和原代CD 4细胞的HIV。为了产生对潜伏期激活高度有效的抗降解ELL 2并控制上游信号传导途径以进一步增强这种作用，我们将鉴定ELL 2中的磷酸化位点和控制ELL 2稳定性和BFEC形成的负责激酶。最后，为了阐明导致ELL 2降解的蛋白水解途径，我们将测试ELL 2的泛素化是否可以被Tat诱导的磷酸化抑制，从而触发蛋白酶体对ELL 2的降解。主要的努力也将指向鉴定对ELL 2特异的泛素化酶，这可能揭示可以被抑制以稳定ELL 2用于有效BFEC形成的靶点。总之，拟议的研究可能有助于开发新的预防性治疗策略，以特异性和有效地根除HIV患者中的潜伏性储库。 公共卫生相关性：潜伏的HIV病毒库是根除感染的主要障碍，因为它们携带沉默的前病毒，一旦治疗中断，它们就会恢复活跃的复制。该提案探索了靶向我们最近发现的对HIV转录至关重要的人类辅助因子的潜力，以重新激活潜伏病毒，然后可以通过高效抗逆转录病毒治疗（HAART）清除。拟议的研究可能使新的连续治疗策略的发展，以具体和有效地消除潜伏的艾滋病毒水库感染患者。

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.