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: 8696832
• 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/8696832
• 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 Methylation; Development; Disease; Drug toxicity; Epigenetic Process; Frequencies; Genes; Genetic Transcription; Genomic DNA; 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，使用针对不同分子靶点的多种组合抗病毒药物，可以在较长时间内实现对病毒复制的最大抑制。然而，只要坚持治疗方案，这种成功就会持续下去。对于绝大多数感染患者来说，停止治疗会导致病毒在数周内反弹，需要长期、终身接受抗逆转录病毒治疗。药物诱导根除HIV感染的障碍是病毒潜伏期。许多研究表明，静息的CD 4 T细胞，主要是那些具有中央记忆功能的细胞，构成了潜伏性HIV感染的主要储存库。由于记忆T细胞具有长寿命，因此预测这种储存库非常稳定，因此不容易通过自然细胞衰变而耗尽。最近在艾滋病毒治疗方面的努力已经转向发展针对病毒储存库和干扰对潜伏感染的控制的策略。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HIV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是有限的。推进这一领域研究的一个主要障碍是在受感染个体的外周循环中发现这种潜伏感染细胞的频率极低（约每百万个CD 4细胞中有1 - 10个）。此外，这种潜伏感染的CD 4细胞不能被分离用于研究，因为它们主要以静止细胞状态存在于组织中，并且不能从未感染的静止T细胞中进行表型区分。这些对潜伏感染的体内和离体研究的障碍使得使用体外细胞模型成为必要。尽管这些模型中的大多数由在永生化的人T细胞系和克隆中抑制HIV复制组成，但近年来已经可以获得具有沉默病毒感染的原代T细胞的连续培养系统。从这些模型产生的结果表明，在病毒转录启动的长末端重复序列（LTR）的各种潜在的分子块。由于所有这些模型都使用了持续循环的T细胞，目前还不清楚这些发现与体内HIV潜伏期的实际生物学状态有多少相关。我们提出了一种独特的方法来研究分子相互作用，调节持续/潜伏的HIV感染，使用一个定义明确的感染，非分裂的静息CD 4 T细胞的原代细胞模型。我们假设保守的表观遗传机制控制HIV前病毒的转录状态并调节病毒潜伏期。工作将集中在两个主要组成部分的表观遗传控制：染色质结构在网站的艾滋病毒整合，并在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）使用选定的HDAC的靶向iRNA敲低，确定组蛋白去乙酰化酶（HDAC）功能对维持HIV潜伏期的贡献; 2）使用具有选定乙酰化和甲基化残基修饰的组蛋白的ChIP，确定与病毒转录的抑制与活性状态相关的特定组蛋白修饰;和3）使用亚硫酸氢盐驱动的甲基胞嘧啶序列分析，确定与病毒转录的抑制与活性状态相关的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HIV潜伏期的分子机制的理解-这是开发新治疗策略的关键一步。