Role of FACT Proteins in Regulating HIV Transcription and Latency

 描述(申请人提供)：尽管HAART成功地阻止了艾滋病患者中HIV-1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。最近发现，潜伏的HIV-1宿主的规模比之前估计的要大得多。因此，寻找能够逆转HIV-1潜伏期的新宿主基因是当务之急。我们最近对调节HIV-1复制的宿主因素的研究证实了(促进染色质转录)蛋白SUPT16H和SSRP1限制HIV-1复制的事实。我们的进一步研究表明，FACT蛋白干扰TAT-LTR的转录活性，FACT蛋白的缺失增强了HIV-1的转录，并促进了潜伏的HIV-1的重新激活。在这个提案中，我们将探索FACT蛋白如何负向调控HIV-1复制的分子机制。目的1：我们的遗传学数据显示，FACT蛋白的缺失显著增强了HIV-1的转录。考虑到蛋白质通常促进转录的事实，这一结果令人惊讶。研究FACT在HIV-1感染环境下的作用是至关重要的 将提供有关可能是HIV-1独有的事实功能的新知识。我们假设可能导致事实抑制活动的两种可能机制：(I)。FACT蛋白的存在可能影响HIV-1转录延伸中P-TEFb的活性；FACT蛋白可能为HIV-1转录处理改变的核小体交换活性。这些假设将通过使用多学科方法的一系列实验进行彻底检验。目的：我们的初步结果表明，SUPT16H可能直接与HIV-1 Tat结合，并募集到LTR启动子。然而，对于这些相互作用的进一步描述，仍然缺乏大量信息。因此，我们将研究FACT与HIV-1TAT-LTR以及其他关键宿主转录因子(PAF1和P-TEFb)相互作用的分子细节。我们将(I)定位SUPT16H的哪个结构域(S)介导其与TAT的直接相互作用；(Ii)确定SUPT16H的LTR招募是否取决于TAT或PAF1；(Iii)评估FACT蛋白对P-TEFb和TAT-LTR相互作用的影响。目的3：如果事实蛋白真的对HIV-1转录产生抑制作用，它们可能通过对HIV-1潜伏期的调节而发挥作用。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蛋白会自发地逆转HIV-1潜伏。为此，我们将在一个原代的CD4+T细胞模型中进一步评估FACT蛋白对HIV-1潜伏期的影响：(I)将确定FACT蛋白在抑制HIV-1转录建立潜伏期中的作用；(Ii)将测量FACT蛋白耗尽对潜伏期HIV-1重新激活的影响。(Iii)将研究FACT蛋白与其他HIV-1转录抑制因子--组蛋白脱乙酰酶(HDAC)的协调。最重要的是，我们期望我们对FACT蛋白的深入功能研究将有助于了解它们在HIV-1复制中的作用，并为针对它们进行HIV-1抗潜伏期治疗提供足够的科学基础。

 DESCRIPTION (provided by applicant): 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 wil 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 P- TEFb 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.