Role of FACT Proteins in Regulating HIV Transcription and Latency

项目摘要：尽管HAART成功阻断了HIV-1在艾滋病患者中的活跃复制，但它 并不能根除病毒。潜伏的HIV-1宿主的存在仍然是治愈的主要障碍。最近， 据发现，潜伏的HIV-1病毒宿主的规模比之前估计的要大得多。因此， 迫切需要寻找能够逆转HIV-1潜伏期的新宿主基因。我们最近的研究 在调节HIV-1复制的宿主因素中，确定了(促进染色质转录)蛋白质， SUPT16H和SSRP1限制HIV-1复制。我们的进一步研究表明，蛋白质会干扰 具有TAT-LTR转录活性，FACT蛋白的耗尽增强了HIV-1的转录和 促进潜伏的HIV-1的重新激活。在这项提议中，我们将探索分子机制如何 蛋白质负向调节HIV-1的复制。目的1：我们的基因数据显示，FACT蛋白的耗尽 显著增强HIV-1转录。这是一个令人惊讶的结果，因为蛋白质通常 促进转录。研究FACT在HIV-1感染情况下的作用是至关重要的，这将对 提供有关HIV-1独有的事实功能的新知识。我们假设有两种可能 可能导致事实压制活动的机制：(1)。FACT蛋白的存在可能影响PTEFb HIV-1转录延伸的活性；(Ii)FACT蛋白可能处理改变的核小体 HIV-1转录的交换活性。这些假说将通过一系列的 使用多学科方法的实验。目的2：我们的初步结果表明，SUPT16H可能 直接与HIV-1Tat结合并募集到LTR启动子。然而，大量的信息仍然缺乏进一步的 这些相互作用的特征。因此，我们将研究FACT与HIV-1相互作用的分子细节 TAT-Ltr以及其他关键的宿主转录因子(PAF1和P-TEFb)。我们将(I)映射哪个域(S) SUPT16H的作用调节其与TAT的直接相互作用；(Ii)确定SUPT16H的LTR招募 取决于TAT或PAF1；(Iii)评估FACT蛋白对P-TEFb和TAT-Ltr相互作用的影响。 目标3：如果事实蛋白真的对HIV-1转录产生抑制作用，它们可能通过这种作用 在调节HIV-1潜伏期方面发挥作用。我们早期使用HIV-1潜伏感染J-LAT细胞的结果证实 RNAi耗尽FACT蛋白会自发逆转HIV-1潜伏期。为了实现这一目标，我们将进一步 评估FACT蛋白对原代CD4+T细胞模型中HIV-1潜伏期的影响：(I)FACT的作用 将确定抑制HIV-1转录以建立潜伏期的蛋白质；(Ii)事实的影响 将测量潜伏期对HIV-1重新激活的蛋白质消耗。(3)FACT蛋白与 其他HIV-1转录抑制因子，组蛋白脱乙酰酶(HDAC)，将被研究。最重要的是，我们 期待我们对FACT蛋白的深入功能研究将有助于理解它们在HIV-1中的作用 并为针对它们进行HIV-1抗潜伏期治疗提供了足够的科学基础。

PROJECT SUMMARY: Although HAART is successful to block active replication of HIV-1 in AIDS patients, it does not eradicate viruses. Presence of latent HIV-1 reservoirs remains a major obstacle to the cure. Recently, it was uncovered that the size of latent HIV-1 reservoirs is much larger than previously estimated. Thus, identification of novel host genes that can be targeted for reverting HIV-1 latency is urgent. Our recent studies of host factors modulating HIV-1 replication identified that FACT (facilitates chromatin transcription) proteins, SUPT16H and SSRP1, restrict HIV-1 replication. Our further studies demonstrated that FACT proteins interfere with TAT-LTR transcriptional activities, and depletion of FACT proteins enhances HIV-1 transcription and facilitates reactivation of latent HIV-1. In this proposal, we will explore molecular mechanisms how FACT proteins negatively regulate HIV-1 replication. Aim 1: Our genetic data showed that depletion of FACT proteins significantly enhance HIV-1 transcription. It was a surprising result considering that FACT proteins generally facilitate transcription. It is critical to study FACT’s function under circumstance of HIV-1 infection, which will provide new knowledge about FACT functions that might be unique for HIV-1. We postulate two possible mechanisms that might lead to FACT suppressive activities: (i). Presence of FACT proteins might affect PTEFb activity in HIV-1 transcriptional elongation; (ii). FACT proteins might process altered nucleosome exchange activity for HIV-1 transcription. These hypotheses will be thoroughly tested by a series of experiments using multidisciplinary approaches. Aim 2: Our preliminary results indicated that SUPT16H might directly bind with HIV-1 TAT and recruit to LTR promoter. However, a lot of information still lacks for further characterization of these interactions. Thus, we will study the molecular details of FACT interactions with HIV-1 TAT-LTR as well as other key host transcriptional factors (PAF1 and P-TEFb). We will (i) map which domain(s) of SUPT16H mediate its direct interaction with TAT; (ii) determine whether LTR recruitment of SUPT16H depends on TAT or PAF1; (iii) evaluate the impact of FACT proteins on P-TEFb and TAT-LTR interactions. Aim 3: If indeed FACT proteins impose an inhibitory effect on HIV-1 transcription, through which they might play a role in regulating HIV-1 latency. Our earlier results using HIV-1 latently infected J-LAT cells confirmed that depletion of FACT proteins by RNAi spontaneously reverts HIV-1 latency. In this aim, we will further evaluate the effects of FACT proteins on HIV-1 latency in a primary CD4+ T cell model: (i) Role of FACT proteins in suppressing HIV-1 transcription for latency establishment will be determined; (ii) Effects of FACT protein depletion on HIV-1 reactivation from latency will be measured. (iii) Coordination of FACT proteins with other HIV-1 transcriptional suppressors, histone deacetylases (HDACs), will be investigated. Above all, we expect that our in-depth functional studies of FACT proteins will shed light in understanding their roles in HIV-1 replication and provide sufficient scientific foundation to target them for HIV-1 anti-latency therapy.