Evaluating the role of the novel apoptosis inhibitor TRAILshort, in maintaining HIV persistence.

评估新型细胞凋亡抑制剂 TRAILshort 在维持 HIV 持久性方面的作用。

• 批准号: 8990167
• 负责人: ANDREW D BADLEY
• 金额: $ 50.57万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990167
• 关键词: Acute; Antiviral Agents; Apoptosis; Apoptosis Inhibitor; Apoptotic; Autologous; B-Lymphocytes; Binding; Biological Assay; CD4 Positive T Lymphocytes; CD8B1 gene; Caspase; Caspase-1; Cell Count; Cell Death; Cell Death Signaling Process; Cell Line; Cell Survival; Cell model; Cell physiology; Cells; Cessation of life; Coculture Techniques; Complex; DNA; DNA-dependent protein kinase; Data; Death Receptor 5; Dendritic Cells; Disease Progression; Dominant-Negative Mutation; Failure; Goals; Granzyme; HIV; HIV Infections; Herpesvirus 1; Homologous Gene; Human; Human Herpesvirus 4; Human Herpesvirus 8; Human Papillomavirus; IL6 gene; Immune; Immune response; Immune system; Immunologic Surveillance; Immunologics; Impairment; In Vitro; Integrase; Interferon-alpha; Interleukin-6; Intervention; Length; Ligand Binding; Ligands; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Names; Natural Killer Cells; Nested PCR; Outer Mitochondrial Membrane; Pathogenesis; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Plasma; Play; Production; Proteins; Proviruses; RNA Splicing; Regulation; Research; Research Proposals; Resistance; Role; Signal Transduction; Stimulus; Subfamily lentivirinae; System; T-Lymphocyte; TNF gene; TNF-related apoptosis-inducing ligand; Testing; Transcript; Tumor Necrosis Factor Ligand Superfamily Member 6; Variant; Viral; Virion; Virus; Virus Diseases; Work; apoptosis inducing factor; base; cancer cell; cell killing; clinically relevant; cohort; cytotoxic; cytotoxicity; death receptor-4; design; genetic approach; human TNFRSF10A protein; immune activation; in vivo; inhibitor/antagonist; innovation; insight; interest; killings; knock-down; macrophage; novel; novel strategies; prevent; public health relevance; purge; reactivation from latency; receptor; response; sensor; small hairpin RNA; tool

 DESCRIPTION (provided by applicant): The interplay between a pathogenic viral infection and the human host reflects a bi-directional interaction of host sensors (e.g. TLR, RIG, etc.) which detect virus infection, host effector mechanisms which eliminate infected cells (e.g. caspase activation, expression of death inducing ligands including Fas Ligand and TRAIL), and viral adaptations which attempt to subvert the host response (e.g. virally encoded apoptosis inhibitors e.g. EBV encoded BALF1, KSHV encoded vFLIP etc.). Experimental elimination of virally encoded apoptosis inhibitors enhances host cell clearance of virally infected cells, and reduces viral persistence - as examples: inhibiting BALF1 prevents EBV persistence, inhibiting LAT inhibits HSV1 persistence, and disrupting E6 or E7 inhibits HPV persistence. HIV infection can be sensed in human cells for example by IFI-16 sensing of accumulated reverse transcripts leading to caspase 1 dependent pyroptosis, or DNA-PK sensing of integrase mediated host DNA "nicking" leading to p53 dependent apoptosis. However these sensing mechanisms do not eradicate all HIV infected cells, and HIV is as a result, persistent. HIV is not known to encode a protein which antagonizes the hosts ability to eradicate infected cells, yet accumulating evidence suggests that HIV infected cells are more resistant to cell death that corresponding uninfected cells, through an as yet undefined mechanism TNF related apoptosis inducing factor (TRAIL) is a molecule whose principal function is as an effector of immune surveillance, and it has been implicated in the pathogenesis of malignancies, as well as viral infections including HIV. Concerning the role of TRAIL in HIV, considerable data indicate that TRAIL is dysregulated during HIV infection in vivo, and there is ample evidence that treatment of cells from ART suppressed HIV- infected patients with exogenous TRAIL, reduces the number of latently infected cells, as measured by undetectable levels of replication competent virus in quantitative co-culture assays. The current research proposal concerns a novel TRAIL splice variant, which we have discovered and named TRAILshort, which is produced during HIV infection in vitro and in vivo. TRAILshort binds to TRAIL receptor 2 (TRAILR2), yet does not transmit a cell death signal, whereas TRAIL binding to TRAILR2 rapidly results in apoptotic death. Moreover, expression of TRAILshort, prevents TRAIL from engaging TRAILR2, demonstrating that TRAILshort acts as a dominant negative inhibitor of TRAIL mediated killing. Because NK cells and CD8+ CTL kill target cells by TRAIL (in addition to other mechanisms such as Granzyme B), it follows that TRAILshort expression reduces both NK cell and CD8 T cell cytotoxicity. Our underlying hypothesis is that TRAILshort production during HIV infection prevents TRAIL dependent HIV clearance mechanisms which allows HIV infected cells to persist. This hypothesis is supported by novel preliminary data included in this application, where shRNA mediated knockdown of TRAILshort does not alter uninfected T cell survival, yet selectively enhances killing of HIV infected cells, thereby reducing HIV replication by 3.5 logs, and decreasing the number of cells containing HIV DNA. The long-term goal of our work is to fully understand and manipulate TRAILshort production in order that normal immune mechanisms can contribute to clearance of virally infected cells, thereby providing an additional tool with which to contribute towards a cure for HIV. This will be fully explored by: (i) studying the regulation of TRAILshort, in order to identify means of inhibiting its production. (ii) Optimize lentivirus knock down of TRAILshort in primary CD4 T cells from HIV infected patients, and test whether HIV reactivation with TRAILshort knockdown, plus or minus co-culture with autologous CD8 T cells, or NK cells, reduces HIV reservoir size ex vivo (iii) Evaluate TRAILshort expression in HIV elite controllers, partial controllers and non-controllers, and determine the association of TRAILshort expression with HIV reservoir size.

 描述（由申请人提供）：病原性病毒感染与人类宿主之间的相互作用反映了宿主传感器（例如TLR、RIG等）的双向相互作用。其检测病毒感染、消除感染细胞的宿主效应机制（例如半胱天冬酶活化、包括Fas配体和TRAIL的死亡诱导配体的表达）和试图破坏宿主应答的病毒适应（例如病毒编码的凋亡抑制剂，例如EBV编码的BALF 1、KSHV编码的vFLIP等）。病毒编码的凋亡抑制剂的实验性消除增强了病毒感染细胞的宿主细胞清除，并降低了病毒持久性-例如：抑制BALF 1防止EBV持久性，抑制LAT抑制HSV 1持久性，破坏E6或E7抑制HPV持久性。HIV感染可以在人细胞中被感测，例如通过IFI-16感测积累的逆转录物，导致半胱天冬酶1依赖性细胞凋亡，或DNA-PK感测整合酶介导的宿主DNA“切口”，导致p53依赖性细胞凋亡。然而，这些传感机制并不能根除所有感染HIV的细胞，因此HIV是持久的。目前还不知道HIV编码拮抗宿主根除感染细胞的能力的蛋白质，但越来越多的证据表明，HIV感染的细胞比相应的未感染细胞更能抵抗细胞死亡，通过一种尚未确定的机制，TNF相关的凋亡诱导因子（TRAIL）是一种分子，其主要功能是作为免疫监视的效应子，并且它与恶性肿瘤以及包括HIV在内的病毒感染的发病机制有关。关于TRAIL在HIV中的作用，相当多的数据表明TRAIL在体内HIV感染期间失调，并且有充分的证据表明，用外源性TRAIL处理来自ART的细胞抑制了HIV感染的患者，减少了潜伏感染细胞的数量，如通过定量共培养测定中不可检测水平的复制能力病毒所测量的。目前的研究计划涉及一种新的TRAIL剪接变体，我们发现并命名为TRAILshort，它在体外和体内HIV感染过程中产生。TRAIL短与TRAIL受体2（TRAILR 2）结合，但不传递细胞死亡信号，而TRAIL与TRAILR 2结合迅速导致凋亡性死亡。此外，TRAIL短的表达阻止TRAIL与TRAILR 2接合，表明TRAIL短充当TRAIL介导的杀伤的显性负性抑制剂。因为NK细胞和CD 8 + CTL通过TRAIL（除了其他机制如颗粒酶B之外）杀死靶细胞，因此TRAIL短表达降低了NK细胞和CD 8 T细胞的细胞毒性。我们的基本假设是，HIV感染过程中TRAIL短的产生阻止了TRAIL依赖的HIV清除机制，这使得HIV感染的细胞持续存在。这一假设得到了本申请中包括的新的初步数据的支持，其中shRNA介导的TRAIL短敲低不改变未感染的T细胞存活，但选择性地增强对HIV感染细胞的杀伤，从而使HIV复制减少3.5个对数，并减少含有HIV DNA的细胞的数量。我们工作的长期目标是充分理解和操纵TRAIL短的生产，以便正常的免疫机制可以有助于清除病毒感染的细胞，从而提供一个额外的工具，有助于治愈艾滋病毒。将通过以下方式充分探讨这一问题：（一）研究TRAIL短的管理，以确定抑制其生产的手段。(ii)优化来自HIV感染患者的原代CD 4 T细胞中的TRAIL短的慢病毒敲低，并测试用TRAIL短敲低的HIV再活化，加上或减去与自体CD 8 T细胞或NK细胞的共培养，是否降低了离体HIV储库大小（iii）评估HIV精英控制者、部分控制者和非控制者中的TRAIL短表达，并确定与HIV感染患者的HIV感染的相关性。 TRAIL短表达与HIV储库大小。