Mode of action of a new Tat HIV-1 inhibitor

新型 Tat HIV-1 抑制剂的作用方式

• 批准号: 9750313
• 负责人: Susana T Valente
• 金额: $ 75.04万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750313
• 关键词: Acute; Aftercare; Antiviral Agents; BLT mice; Binding; Biological Assay; Blood; CD4 Positive T Lymphocytes; Capsid; Cells; Clinical; Collaborations; DNA; Deposition; Dose; Drug resistance; Epigenetic Process; Feedback; Genetic; Genetic Transcription; Genome; Goals; HIV; HIV Infections; HIV-1; Human; Immune system; In Vitro; Individual; Interruption; Lead; Mediating; Messenger RNA; Modification; Molecular; Monitor; Mus; Mutation; NF-kappa B; Nature; Nucleosomes; Outcome; Phase; Positioning Attribute; Production; Property; RNA; Regimen; Residual state; Resistance; Rest; Sum; T-Cell Depletion; T-Lymphocyte; Therapeutic; Time; Tissues; Transactivation; Transcript; Transcription Initiation Site; Transcriptional Regulation; Translating; Universities; Viral; Viral Proteins; Viral reservoir; Viremia; Virus; Work; antiretroviral therapy; base; cortistatin; fitness; gene repression; humanized mouse; immune clearance; in vivo; inhibitor/antagonist; insight; memory CD4 T lymphocyte; mouse model; novel; prevent; promoter; prophylactic; reactivation from latency; recruit; tat Protein; treatment duration; viral RNA; viral rebound; viral resistance

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.

HIV-1达特激活病毒转录，有限的塔特反式激活与潜伏期建立相关。我们基于Tat抑制剂二脱氢皮质抑素A（dCA）的性质假设了一种“阻断和锁定”功能性治疗方法。HIV-1转录抑制剂可以阻断抗逆转录病毒治疗期间的持续病毒血症 (ART)将HIV启动子锁定在持续的潜伏期。我们在从病毒血症个体分离的人CD 4 +T细胞中研究了这一假设。将dCA与ART联合使用可加速HIV-1抑制，并防止治疗中断后病毒反弹，即使在强烈的细胞活化期间也是如此。我们表明，dCA介导 通过增加核小体-1处的核小体占据，限制RNAPII募集至HIV-1启动子，从而导致表观遗传沉默。在HIV潜伏期和持续性的骨髓-肝-胸腺（BLT）小鼠模型中研究了dCA的疗效。向ART抑制的小鼠中添加dCA全身性地减少组织中的病毒mRNA。 此外，dCA显著延迟并降低治疗中断后的病毒反弹水平。总之，这项工作表明了“封锁和锁定”治疗策略的潜力。达特抑制剂不同于任何其他HIV抑制剂，因为治疗持续时间会影响结果，因为Tat-TAR活性的反馈性质，并且因为沉积在HIV-1启动子处的表观遗传标记会随着时间的推移而累积。我们假设，随着时间的推移，转录抑制可以被推过一定的阈值，在该阈值下，病毒从潜伏期重新激活是非常难以克服的，从而将HIV阻断并锁定在持续的潜伏期。dCA的相加活性也支持了将达特抑制剂加入一线抗肿瘤药物中的观点。 治疗可能导致更快的抑制并潜在地减小已建立的储库的大小。从对早期ART治疗的个体的研究中发现，较小的储库大小直接转化为更好的病毒控制（14）。研究了体外病毒对dCA耐药性的遗传屏障，出乎意料但不太令人惊讶的是，未发现达特和TAR突变，因为这些突变非常保守。相反，耐dCA的病毒发展了非常高的Tat独立的基础转录。我们发现了一种突变的组合， 增加基础转录活性的LTR启动子，以及增加NF-κB活性的Nef和Vpr修饰。我们假设这些病毒可能不会在体内发展，我们认为它们增加的转录适应性和无法控制进入潜伏期最终可能是有害的，导致高细胞病变效应和/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治疗时间与残留病毒RNA产生减少之间的关系，以及如何在治疗中断后延迟病毒反弹; 2）研究dCA作为一线治疗对急性期治疗期间已建立的病毒库大小的影响; 3）研究体内病毒对dCA耐药的机制。