Role of FACT Proteins in Regulating HIV Transcription and Latency

FACT 蛋白在调节 HIV 转录和潜伏期中的作用

基本信息

批准号：
9139906
负责人：
Jian Zhu
金额：
$ 29.56万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-15 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9139906
关键词：
AIDS/HIV problem Acquired Immunodeficiency Syndrome Affect Anti-Retroviral Agents Back Binding CD4 Positive T Lymphocytes Cell model Cells Chromatin Complex DNA Polymerase II Data Deposition Disease Foundations Genes Genetic Genetic Transcription Goals HIV HIV-1 Health Highly Active Antiretroviral Therapy Histones In Vitro Infection Integration Host Factors Investigation Knowledge Lead Light Maps Measures Mediating Molecular Molecular Chaperones Nucleosomes Patients Play Positive Transcriptional Elongation Factor B Process Proteins Proviruses RNA Interference Recruitment Activity Regimen Residual state Rest Role Series Small Interfering RNA Structure T-Lymphocyte Testing Viral Viral Load result Viremia Virus Virus Latency antiretroviral therapy base dimer follow-up genome-wide improved in vivo interdisciplinary approach interest memory CD4 T lymphocyte novel prevent promoter purge reactivation from latency research study

项目摘要

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.

描述（由适用提供）：尽管Haart成功地阻止了AIDS患者中HIV-1的主动复制，但它不是放射性病毒。潜在的HIV-1水库的存在仍然是治愈的主要障碍。最近，发现潜在的HIV-1储层的大小比以前估计的要大得多。这是紧迫地鉴定出可用于恢复HIV-1潜伏期的新型宿主基因。我们对调节HIV-1复制的宿主因子的最新研究确定了事实（促进染色质转录）蛋白SUPT16H和SSRP1限制了HIV-1复制。我们的进一步研究表明，事实蛋白会干扰TAT-LTR转录活性，而事实蛋白的耗竭增强了HIV-1转录，并促进潜在HIV-1的重新激活。在此提案中，我们将探索分子机制，事实蛋白如何负调节HIV-1复制。目标1：我们的遗传数据表明，事实蛋白的耗竭可显着增强HIV-1转录。考虑到事实蛋白通常会促进转录，这是一个令人惊讶的结果。在HIV-1感染情况下研究事实的功能至关重要，这将提供有关HIV-1可能是唯一的事实功能的新知识。我们假设两种可能导致事实抑制活动的机制：（i）。事实蛋白的存在可能会影响HIV-1转录伸长的P-TEFB活性。（ii）。事实蛋白可能会处理HIV-1转录的核小体交换活性改变。这些假设将通过使用多学科方法的一系列实验对这些假设进行彻底检验。 AIM 2：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。但是，对于这些相互作用的进一步表征仍然缺乏许多信息。这是，我们将研究与HIV-1 TAT-LTR以及其他关键宿主转录因子（PAF1和P-TEFB）的事实相互作用的分子细节。我们将（i）映射哪个SUPT16H的域介导了与TAT的直接相互作用；（ii）确定SUPT16H的LTR募集是否取决于TAT或PAF1；（iii）评估事实蛋白对P-TEFB和TAT-LTR相互作用的影响。目标3：如果确实事实蛋白会对HIV-1转录产生抑制作用，它们可能在调节HIV-1潜伏期中发挥作用。我们使用HIV-1潜在感染的J-LAT细胞的早期结果证实，RNAi赞助的事实蛋白的耗竭会恢复HIV-1潜伏期。在此目标中，我们将进一步评估事实蛋白对主要CD4+ T细胞模型中HIV-1潜伏期的影响：（i）将确定事实蛋白在抑制HIV-1转录中的作用；（ii）将测量蛋白质耗竭对潜伏期HIV-1重新激活的影响。（iii）将研究事实蛋白与其他HIV-1转录补充剂，组蛋白脱乙酰基酶（HDACS）的协调。最重要的是，我们期望我们对事实蛋白的深入功能研究将揭示其在HIV-1复制中的作用，并提供足够的科学基础，以将其瞄准HIV-1抗延迟治疗。