Inhibition of heat shock protein 90 for sustained remission of HIV from persistent tissue reservoirs

项目总结 持续感染HIV的静息CD4+T细胞在组织和器官储存库中可能保持未被检测到，尽管 几十年成功的抗逆转录病毒治疗(ART)。静息的CD4+T细胞缺乏基因活性使 具有复制能力的集成前病毒可以无限期地持续存在。HIV在活化的CD4+T细胞中的复制 细胞病变，受感染的细胞被免疫系统清除。然而，一小部分艾滋病毒感染者 激活的CD4+T细胞恢复到静止的G0表型，在组织储存库中仍未被检测到。这些艾滋病毒- 在ART中断的患者中，感染的静息细胞可以很容易地被激活，并反弹病毒血症 来源于持续感染艾滋病毒的组织储存库。 我的实验室正在进行的一个项目是关于热休克蛋白90(Hsp90)在HIV中的重要作用 复制。我们已经证明，温和的热休克(39.5°C)加速了慢性感染患者的HIV转录 T细胞株，并将艾滋病毒在原代人类细胞中的复制增加30倍。加速HIV转录 与39.5°C下细胞Hsp90活性增加相吻合。Hsp90负责激活细胞 转录因子，而Hsp90抑制剂17-AAG通过核因子-kB，NFAT， 和STAT5受体转录因子。此外，39.5℃可重新激活ART抑制的无核小鼠中的HIV复制 HIV感染患者样本和从完全抑制的人源化小鼠中分离出的人类静息CD4+T细胞。 此外，我们还表明，更有效的下一代Hsp90抑制剂具有更强的抗HIV活性 高度有利的治疗排名，临床艾滋病毒亚型在39.5℃时显示病毒转录增加 在原代人类T细胞和巨噬细胞中。 最重要的是，抑制Hsp90可以防止HIV在完全受抑制的人源化小鼠中反弹，我们发现 小鼠脾中持续感染艾滋病毒的人类细胞。两种不同的Hsp90抑制剂可阻断HIV 在这个持久性组织库中转录，并在停药后持续艾滋病毒缓解长达11周。 此外，我们现在有证据表明，在脾、脑和肺的水库中持续的艾滋病毒感染是成立的。 接种后不久，会产生传染性病毒，尽管ART具有很强的效力。人类感染HIV的T细胞 巨噬细胞也存在于小鼠的肝脏、骨髓和生殖道中。 我们假设，在39.5℃时，Hsp90活性增加会激活宿主转录因子，这些转录因子是 艾滋病毒转录。这一假设将在以下具体目标中得到解决：1)确定基因 热休克激活的HIV感染者静息CD4+T细胞的表达谱2)特异性Hsp90的鉴定 调节HIV在静止的CD4+T细胞中持续存在的蛋白质复合体，以及3)持续HIV的特征 体内蓄水池并调查反跳性病毒血症的来源。 我们预计这些研究将有助于更好地了解Hsp90是如何调控HIV转录的，以及 我们在体内的结果将为深入了解持续性组织储存库的反跳性病毒血症的性质提供依据。

PROJECT SUMMARY Persistent HIV-infected resting CD4+ T cells can remain undetected in tissue and organ reservoirs despite decades of successful antiretroviral therapy (ART). The lack of gene activity in resting CD4+ T cells enables the integrated replication-competent provirus to persist indefinitely. HIV replication in activated CD4+ T cells is cytopathic, and infected cells are cleared by the immune system. However, a small percentage of HIV-infected activated CD4+ T cells revert to the resting G0 phenotype and remain undetected in tissue reservoirs. These HIV- infected resting cells can be readily activated, and rebound viremia in patients with interrupted ART invariably originates from persistent HIV-infected tissue reservoirs. An ongoing project in my laboratory is focused on the essential role of heat shock protein 90 (Hsp90) in HIV replication. We have shown that mild heat shock (39.5°C) accelerates HIV transcription in chronically infected T-cell lines and increases HIV replication up to 30-fold in primary human cells. Accelerated HIV transcription coincides with increased cellular Hsp90 activity at 39.5°C. Hsp90 is responsible for activating cellular transcription factors, and the Hsp90 inhibitor 17-AAG significantly reduces gene expression by the NF-kB, NFAT, and STAT5 host transcription factors. Further, 39.5°C reactivates HIV replication in ART-suppressed aviremic HIV-infected patient samples and in human resting CD4+ T cells isolated from fully suppressed humanized mice. Further, we show that a more potent next-generation Hsp90 inhibitor has even greater anti-HIV activity with a highly favorable therapeutic ranking and that clinical HIV subtypes display increased viral transcription at 39.5°C in primary human T cells and macrophages. Most importantly, Hsp90 inhibition prevents HIV rebound in fully suppressed humanized mice, and we identified persistent HIV-infected human cells in the mouse spleen. Two different Hsp90 inhibitors blocked HIV transcription in this persistent tissue reservoir and sustained HIV remission up to 11 weeks after drug cessation. Further, we now have evidence that persistent HIV infection in spleen, brain, and lung reservoirs is established soon after inoculation, which produces infectious virus despite highly potent ART. HIV-infected human T cells and macrophages are also found in the mouse liver, bone marrow, and reproductive tract. We hypothesize that increased Hsp90 activity at 39.5°C activates host transcription factors that are essential for HIV transcription. This hypothesis will be addressed in the following Specific Aims: 1) Determine the gene expression profile of heat-shock activated HIV-infected human resting CD4+ T cells, 2) Identify specific Hsp90 protein complexes that regulate HIV persistence in resting CD4+ T cells, and 3) Characterize persistent HIV reservoirs in vivo and investigate the source of rebound viremia. We anticipate these studies will lead to a better understanding of how Hsp90 regulates HIV transcription, and our in vivo results will provide insight into the nature of rebound viremia from persistent tissue reservoirs.