Role of FACT Proteins in Regulating HIV Transcription and Latency

 描述（由申请人提供）：尽管HAART成功地阻断了艾滋病患者体内HIV-1的活跃复制，但它不能根除病毒。潜伏的HIV-1储库的存在仍然是治愈的主要障碍。最近，人们发现潜伏的HIV-1病毒库的规模比以前估计的要大得多。因此，鉴定新的宿主基因，可以有针对性地逆转HIV-1潜伏期是迫切的。我们最近对调节HIV-1复制的宿主因子的研究发现，FACT（促进染色质转录）蛋白SUPT 16 H和SSRP 1限制HIV-1复制。我们的进一步研究表明，FACT蛋白干扰TAT-LTR转录活性，FACT蛋白的缺失增强HIV-1转录并促进潜伏HIV-1的再激活。在本研究中，我们将探讨FACT蛋白负调控HIV-1复制的分子机制。目的1：我们的遗传数据表明，FACT蛋白的缺失显着增强HIV-1的转录。考虑到FACT蛋白通常促进转录，这是一个令人惊讶的结果。研究FACT在HIV-1感染情况下的功能至关重要， 将提供关于FACT功能的新知识，这些功能可能是HIV-1所独有的。我们假设可能导致FACT抑制活性的两种可能机制：（i）。FACT蛋白的存在可能影响P-TEFb在HIV-1转录延伸中的活性;（ii）. FACT蛋白可能改变HIV-1转录的核小体交换活性。这些假设将通过使用多学科方法的一系列实验进行彻底测试。目的2：我们的初步研究结果表明SUPT 16 H可能直接与HIV-1达特结合并募集到LTR启动子。然而，许多信息仍然缺乏进一步表征这些相互作用。因此，我们将研究FACT与HIV-1 TAT-LTR以及其他关键宿主转录因子（PAF 1和P-TEFb）相互作用的分子细节。我们将（i）绘制SUPT 16 H的哪个或哪些结构域介导其与达特的直接相互作用;（ii）确定SUPT 16 H的LTR募集是否依赖于达特或PAF 1;（iii）评价FACT蛋白对P-TEFb和TAT-LTR相互作用的影响。目的3：如果FACT蛋白确实对HIV-1转录产生抑制作用，那么它们可能在调节HIV-1潜伏期中发挥作用。我们使用HIV-1潜伏感染的J-LAT细胞的早期结果证实，通过RNAi消耗FACT蛋白自发地逆转HIV-1潜伏期。为此，我们将在原代CD 4 + T细胞模型中进一步评价FACT蛋白对HIV-1潜伏期的影响：（i）将确定FACT蛋白在抑制HIV-1转录以建立潜伏期中的作用;（ii）将测量FACT蛋白耗竭对HIV-1从潜伏期再激活的影响。(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）的协调作用。最重要的是，我们希望我们对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，并为靶向它们进行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.