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：
8920866
负责人：
Jian Zhu
金额：
$ 29.55万
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/8920866
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 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 public health relevance 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（由申请人提供）：尽管HAART 成功地阻止了艾滋病患者体内HIV-1 的活跃复制，但它并不能根除潜伏HIV-1 病毒库的存在，这仍然是治疗的主要障碍。潜在的 HIV-1 储存库的大小比之前估计的要大得多，因此，迫切需要鉴定可逆转 HIV-1 潜伏期的新宿主基因。 FACT（促进染色质转录）蛋白 SUPT16H 和 SSRP1 限制 HIV-1 复制，FACT 蛋白干扰 TAT-LTR 转录活性，并且 FACT 蛋白的耗尽增强 HIV-1 转录并促进潜伏 HIV-1 的重新激活。 1. 在本提案中，我们将探讨 FACT 蛋白如何负向调节 HIV-1 复制的分子机制目标 1：我们的遗传数据表明，FACT 蛋白的消耗显着增强。 HIV-1转录是一个令人惊讶的结果，因为FACT蛋白通常促进转录，因此研究FACT在HIV-1感染情况下的功能至关重要。将提供有关 HIV-1 可能独特的 FACT 功能的新知识我们假设可能导致 FACT 抑制活性的两种可能机制：（i）FACT 蛋白的存在可能影响 HIV-1 转录延伸中的 P-TEFb 活性； (ii).FACT 蛋白可能会改变 HIV-1 转录的核小体交换活性。目标 2：我们的初步结果表明 SUPT16H 可能会发生变化。然而，仍缺乏大量信息来进一步表征这些相互作用，因此，我们将研究 FACT 与 HIV-1 TAT-LTR 以及其他相互作用的分子细节。我们将 (i) 绘制 SUPT16H 的哪些结构域介导其与 TAT 的直接相互作用；(ii) 确定 SUPT16H 的 LTR 募集是否依赖于 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 FACT 蛋白确实对 HIV-1 转录产生抑制作用，那么它们可能通过这种作用发挥调节 HIV-1 的作用。我们早期使用 HIV-1 潜伏感染的 J-LAT 细胞的结果证实，RNAi 消除 FACT 蛋白会自发恢复 HIV-1 潜伏期。为此，我们将进一步评估 FACT 蛋白对潜伏期的影响。原代 CD4+ T 细胞模型中的 HIV-1 潜伏期：(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 蛋白消耗对 HIV-1 从潜伏期重新激活的影响； (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) 的协调作用。最重要的是，我们期望对 FACT 蛋白的深入功能研究将有所启发。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。