Control of Latent/Persistent HIV Infection in Primary CD4 T Cells

控制原代 CD4 T 细胞中的潜伏/持续 HIV 感染

• 批准号: 8280133
• 负责人: CELSA A SPINA
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280133
• 关键词: Acetylation; Adherence; Adult; Antiviral Agents; Area; Beryllium; Binding; Blood Circulation; CD4 Positive T Lymphocytes; Caring; Cell Extracts; Cell Line; Cell model; Cells; Cessation of life; Chromatin Structure; Chronic; DNA; DNA Methylation; Development; Disease; Drug toxicity; Epigenetic Process; Frequencies; Genes; Genetic Transcription; Genomics; HIV; HIV Infections; HIV Long Terminal Repeat; Healthcare Systems; Histone Acetylation; Histone Deacetylase; Histones; Human; In Vitro; Individual; Infection; Interphase Cell; Knowledge; Lead; Long Terminal Repeats; Longevity; Maintenance; Memory; Methylation; Modeling; Modification; Molecular; Molecular Target; Molecular Weight; Morbidity - disease rate; Patients; Pattern; Peripheral; Pharmaceutical Preparations; Pharmacotherapy; Post-Translational Protein Processing; Promoter Regions; Provider; Proviruses; RNA; Reagent; Research; Residual state; Rest; Sequence Analysis; Site; System; T memory cell; T-Lymphocyte; Terminal Repeat Sequences; Therapeutic; Time; Tissues; Transcriptional Regulation; Treatment Protocols; Viral; Virus Diseases; Virus Latency; Work; antiretroviral therapy; bisulfite; body system; chromatin immunoprecipitation; design; histone modification; in vivo; innovation; latent infection; mortality; success; therapy design; treatment strategy

DESCRIPTION (provided by applicant): Current treatment for HIV infection, using multiple combined antiviral drugs to different molecular targets, can achieve maximal suppression of viral replication for extended periods. Nevertheless, this success lasts only as long as adherence to the treatment regimen is maintained. For the vast majority of infected patients, discontinuation of treatment results in viral rebound within weeks, requiring tchronic, lifelong administration of antiretroviral therapy. The obstacle to drug induced eradication of HIV infection is viral latency. Many studies indicate that resting CD4 T cells, primarily those with central memory function, compose a major reservoir for latent HIV infection. Because memory T cells have long life spans, such reservoirs are predicted to be extremely stable and therefore, not amenable to depletion through natural cellular decay. Recent efforts in HIV treatment have turned towards the development of strategies to target the viral reservoir and interfere with control of latent infection. However, before effective therapeutic approaches can be designed, a better understanding of the basic biologic mechanisms underlying the establishment and maintenance of persistent, nonproductive HIV infection is needed. Our knowledge of the interactions between viral and cellular elements that are required to maintain HIV latency is limited at present. A major hurdle to advancing research in this area is the extremely low frequency at which such latently infected cells are found in the peripheral circulation of infected individuals (approximately 1 - 10 per million CD4 cells). In addition, such latently infected CD4 cells cannot be isolated for study, because they persist mainly in tissues in a quiescent cell state and cannot be phenotypically discerned from uninfected resting T cells. These impediments to in vivo and ex vivo study of latent infection have necessitated the use of in vitro cell models. Although the majority of these models have consisted of repressed HIV replication in immortalized human T cell lines and clones, continuous culture systems of primary T cells with silenced viral infection have become available in recent years. Results generated from these models indicate various potential molecular blocks in viral transcription initiated from the long terminal repeat (LTR). Because all of these models use continuously cycling T cells, it remains unclear how much of the findings are relevant to the actual biologic state of HIV latency in vivo. We propose a unique approach to study molecular interactions that regulate persistent/latent HIV infection using a well-defined primary cell model of infected, non-dividing resting CD4 T cells. We hypothesize that conserved epigenetic mechanisms control the transcriptional state of the HIV provirus and regulate viral latency. Work will focus on two major components of epigenetic control: chromatin structure at the site of HIV integration, and sequence specific DNA methylation in the LTR promoter region. The specific aims of the project are to: 1) determine the contribution of histone deacetylase (HDAC) function to the maintenance of HIV latency, using targeted iRNA knockdown of selected HDACs; 2) determine specific histone modifications associated with a repressed vs. active state of viral transcription, using ChIP of histones with select acetylated and methylated residue modifications; and 3) determine HIV LTR sequence specific methylation patterns associated with a repressed vs. active state of viral transcription, using bisulfite driven methylcytosine sequence analysis. Knowledge derived from these studies will add significantly to our understanding of the molecular mechanisms controlling HIV latency --- which is a critical step in the path to developing new treatment strategies.

描述(由申请人提供)： 目前对HIV感染的治疗方法是，针对不同的分子靶点使用多种联合抗病毒药物，可以在较长时间内最大限度地抑制病毒复制。然而，这种成功只有在坚持治疗方案的情况下才能持续。对于绝大多数感染者来说，停止治疗会导致病毒在几周内反弹，需要长期、终生接受抗逆转录病毒治疗。药物导致的艾滋病毒感染根除的障碍是病毒潜伏期。许多研究表明，静息的CD4T细胞，主要是那些具有中央记忆功能的细胞，构成了潜伏的HIV感染的主要储备库。因为记忆T细胞的寿命很长，所以预测这种储存库非常稳定，因此不会因为细胞的自然腐烂而耗尽。最近在艾滋病毒治疗方面的努力已转向制定针对病毒库和干扰潜伏感染控制的战略。然而，在设计有效的治疗方法之前，需要更好地了解建立和维持持续性、非生产性艾滋病毒感染的基本生物学机制。目前，我们对维持HIV潜伏期所需的病毒和细胞成分之间的相互作用的了解有限。推进这一领域研究的一个主要障碍是在感染者的外周循环中发现这种潜伏感染细胞的频率极低(大约每百万个CD4细胞中有1-10个)。此外，这种潜伏感染的CD4细胞不能被分离出来进行研究，因为它们主要存在于处于静止细胞状态的组织中，不能从未感染的静止T细胞中区分出来。这些阻碍了体内和体外潜伏感染的研究，因此有必要使用体外细胞模型。虽然这些模型中的大多数都是在永生化的人类T细胞系和克隆中抑制HIV的复制，但近年来已经有了沉默病毒感染的原代T细胞的连续培养系统。从这些模型中产生的结果表明，在病毒转录中，由长末端重复序列(LTR)启动的各种潜在的分子阻断。由于所有这些模型都使用持续循环的T细胞，目前尚不清楚这些发现在多大程度上与体内HIV潜伏期的实际生物状态有关。我们提出了一种独特的方法来研究调节持续/潜伏的HIV感染的分子相互作用，使用一个明确定义的感染的、未分裂的静止CD4T细胞的原代细胞模型。我们假设，保守的表观遗传机制控制着HIV前病毒的转录状态，并调节病毒潜伏期。工作将集中在表观遗传控制的两个主要组成部分：HIV整合部位的染色质结构，以及LTR启动子区域的序列特异性DNA甲基化。该项目的具体目标是：1)通过靶向敲除选定的组蛋白脱乙酰酶(HDAC)，确定组蛋白脱乙酰酶(HDAC)功能对维持HIV潜伏时间的贡献；2)使用带有选择性乙酰化和甲基化残基的组蛋白芯片，确定与病毒转录抑制状态和活跃状态相关的特定组蛋白修饰；以及3)使用亚硫酸氢盐驱动的甲基胞嘧啶序列分析，确定与病毒转录抑制状态和活跃状态相关的HIV LTR序列特异性甲基化模式。从这些研究中获得的知识将大大增加我们对控制艾滋病毒潜伏期的分子机制的了解-这是开发新的治疗策略的关键一步。