Role of FACT Proteins in Regulating HIV Transcription and Latency

项目总结：尽管HAART成功地阻断了艾滋病患者体内HIV-1的活跃复制， 并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， 发现潜伏的HIV-1储存库的规模比以前估计的要大得多。因此，在本发明中， 鉴定能够逆转HIV-1潜伏期的新宿主基因是当务之急。我们最近的研究 调节HIV-1复制的宿主因子鉴定出FACT（促进染色质转录）蛋白， SUPT 16 H和SSRP 1限制HIV-1复制。我们进一步的研究表明，FACT蛋白干扰 具有TAT-LTR转录活性，FACT蛋白的缺失增强HIV-1转录， 促进潜伏的HIV-1的重新激活。在这个提议中，我们将探讨FACT如何影响 蛋白质负调节HIV-1复制。目的1：我们的遗传数据表明，FACT蛋白的缺失 显著增强HIV-1转录。这是一个令人惊讶的结果，因为FACT蛋白通常 便于转录。研究FACT在HIV-1感染情况下的功能，对HIV-1感染的预防和治疗具有重要意义。 提供了关于FACT功能的新知识，这些功能可能是HIV-1所独有的。我们假设两种可能 可能导致FACT抑制活性的机制：（i）。FACT蛋白的存在可能影响PTEFb 在HIV-1转录延伸中的活性;（ii）. FACT蛋白可能加工改变的核小体 HIV-1转录的交换活性。这些假设将通过一系列的 使用多学科方法进行实验。目的2：我们的初步研究结果表明SUPT 16 H可能 直接与HIV-1 TAT结合并募集至LTR启动子。然而，许多信息仍然缺乏进一步的研究。 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以及其他关键宿主转录因子（PAF1和P-TEFb）。我们将（i）映射哪个域（s） SUPT 16 H介导其与TAT的直接相互作用;（ii）确定SUPT 16 H的LTR募集是否 取决于TAT或PAF 1;（iii）评估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转录具有抑制作用，那么它们可能 在调节HIV-1潜伏期中发挥作用。我们早期使用HIV-1潜伏感染的J-LAT细胞的结果证实了 通过RNAi消除FACT蛋白自发地逆转HIV-1潜伏期。为此，我们将进一步 在原代CD4 + T细胞模型中评价FACT蛋白对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）FACT的影响 将测量HIV-1从潜伏期再激活时的蛋白质消耗。(iii)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 我希望我们对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 复制，并提供足够的科学基础，针对他们的HIV-1抗潜伏治疗。

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.