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

项目摘要 持续的HIV感染的静息CD 4 + T细胞在组织和器官储库中仍然未被检测到， 抗逆转录病毒治疗（ART）的成功。静息CD 4 + T细胞中基因活性的缺乏使其成为可能。 整合的有复制能力的前病毒无限期地存在。HIV在活化的CD 4 + T细胞中的复制 细胞病变和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 活化的CD 4 + T细胞回复到静止的G 0表型，并且在组织储库中保持未被检测到。这些艾滋病毒- 受感染的静息细胞很容易被激活，在中断ART的患者中， 来源于持续的HIV感染组织库。 我实验室正在进行的一个项目集中在热休克蛋白90（Hsp 90）在艾滋病毒中的重要作用。 复制的我们已经证明，轻度热休克（39.5°C）加速了慢性感染者的HIV转录， T细胞系，并增加艾滋病毒复制高达30倍，在原代人类细胞。加速HIV转录 与在39.5°C下增加的细胞Hsp 90活性一致。热休克蛋白90负责激活细胞 转录因子，Hsp 90抑制剂17-AAG显著降低NF-kB，NFAT， 和STAT 5宿主转录因子。此外，39.5°C在ART抑制的病毒血症中重新激活HIV复制。 HIV感染的患者样品和从完全抑制的人源化小鼠中分离的人静息CD 4 + T细胞。 此外，我们发现，更有效的下一代Hsp 90抑制剂具有更大的抗HIV活性， 在39.5 ℃下，临床HIV亚型显示出病毒转录增加 在原代人类T细胞和巨噬细胞中。 最重要的是，Hsp 90抑制可以防止HIV在完全抑制的人源化小鼠中反弹，我们发现， 小鼠脾脏中持续存在的HIV感染细胞。两种不同的Hsp 90抑制剂阻断HIV 在这个持久的组织库中转录，并在停药后持续HIV缓解长达11周。 此外，我们现在有证据表明，持续的艾滋病毒感染的脾脏，脑，肺水库是建立 接种后不久，尽管有高效力的ART，HIV感染的人类T细胞， 在小鼠肝脏、骨髓和生殖道中也发现了巨噬细胞。 我们推测，在39.5°C下增加的Hsp 90活性激活了宿主转录因子，这些转录因子对于 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感染的人静息CD 4 + T细胞的表达谱，2）鉴定特异性Hsp 90 调节HIV在静息CD 4 + T细胞中持续存在的蛋白质复合物，和3）表征持续存在的HIV 在体内的水库和调查反弹病毒血症的来源。 我们预计这些研究将有助于更好地了解Hsp 90如何调节HIV转录， 我们的体内结果将提供对来自持久组织储库的反弹病毒血症的性质的深入了解。

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.