Mode of action of a new Tat HIV-1 inhibitor

HIV-1TAT激活病毒转录，有限的TAT反式激活与潜伏期的建立相关。基于TAT抑制剂二氢皮质抑素A(DCA)的性质，我们推测了一种“阻断加锁定”的功能性治疗方法。HIV-1转录抑制剂可阻断抗逆转录病毒治疗期间持续的病毒血症 (ART)，将HIV启动子锁定在持续潜伏期。我们在从无氧血症患者分离的人的CD4+T细胞中研究了这一假说。将DCA与ART结合可加速HIV-1抑制，并防止病毒在治疗中断后反弹，即使在细胞强烈激活的情况下也是如此。我们证明了DCA在 通过增加核小体-1的核小体占有率，限制RNAPII招募到HIV-1启动子，使表观遗传沉默。在小鼠骨髓-肝脏-胸腺(BLT)HIV潜伏期和持久性模型上研究了DCA的疗效。在ART抑制的小鼠中加入DCA可系统性地减少组织中的病毒mRNA。 此外，DCA在治疗中断时显著延迟和降低病毒反弹水平。总而言之，这项工作展示了“阻止和锁定”治愈策略的潜力。TAT抑制剂不同于任何其他HIV抑制剂，因为治疗的持续时间会影响结果，因为TAT-TAR活性的反馈性质，以及沉积在HIV-1启动子上的表观遗传标记随着时间的推移而积累。我们假设，随着时间的推移，转录抑制可能会被推到某个阈值以上，在这个阈值上，病毒从潜伏期重新激活是极其困难的，从而阻止并锁定艾滋病毒进入持续的潜伏期。DCA的加性活性也支持将TAT抑制剂添加到一线的概念 处理可能导致更快的抑制，并有可能缩小已建立的储集层的大小。从对早期抗逆转录病毒治疗的个体研究中发现，较小的存储容量直接转化为更好的病毒控制(14)。在体外研究了病毒对DCA抗性的遗传障碍，出乎意料但并不太令人惊讶的是，没有发现Tat和TAR的突变，因为它们是极其保守的。相反，对DCA耐药的病毒产生了非常高的TAT非依赖性基础转录。我们鉴定出了一种突变组合 LtR启动子，增加基础转录活性，以及Nef和Vpr的修饰，增加NF-κB的活性。我们假设这些病毒可能不会在体内发展，我们认为它们转录适合性的提高和无法控制进入潜伏期最终可能是有害的，导致高度的细胞病变效应和/或免疫系统的清除。 展望未来，我们有三个主要目标：1)使用BLT小鼠了解DCA治疗时间和残留病毒RNA产量减少之间的关系，以及这如何转化为延迟治疗中断后病毒的反弹；2)研究DCA作为急性期治疗期间一线治疗对已建立的病毒库大小的影响；以及3)研究病毒对DCA的体内抗药性的机制。

HIV-1 Tat activates viral transcription and limited Tat-transactivation correlates with latency establishment. We postulated a “block-and-lock” functional cure approach based on properties of the Tat-inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increased nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether this work demonstrates the potential of “block-and-lock” cure strategies. A Tat inhibitor is unlike any other HIV inhibitor, as duration of treatment impacts the outcome, because of the feedback nature of the Tat-TAR activity and because epigenetic marks deposited at the HIV-1 promoter accrue over time. We hypothesized that over time transcriptional repression could be pushed past a certain threshold where viral reactivation from latency is extremely difficult to overcome, blocking-and-locking HIV into sustained latency. The additive activity of dCA also supports the notion that adding Tat inhibitors to front-line treatment might lead to faster suppression and potentially reduce the size of the established reservoir. It is emerging from studies of individuals on very early ART treatment that a smaller reservoir size directly translates into better viral control (14). The genetic barrier to viral resistance to dCA in vitro was investigated, and unexpectedly but not too surprising, mutations in Tat and TAR were not found, since these are extremely conserved. Instead, viruses resistant to dCA developed very high Tat-independent basal transcription. We identified a combination of mutation in the LTR promoter that increased basal transcriptional activity, and modifications in Nef and Vpr that increased NF-κB activity. We hypothesize these viruses may not develop in vivo, we reason their increased transcription fitness and inability to control entry into latency may ultimately be detrimental, leading to high cytopathic effects and/or clearance by the immune system. Looking ahead we have three main goals: 1) using BLT mice to understand the relationship between dCA treatment time and reductions in residual viral RNA production and how that translates in delaying viral rebound after treatment interruption; 2) study the impact of dCA as front-line therapy on the size of the established viral reservoir during acute phase treatment; and 3) study mechanisms of viral resistance to dCA in vivo.