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检测病毒感染的宿主传感器（例如TLR、RIG等）和消除受感染细胞的宿主效应器机制（例如病毒）的双向相互作用。 caspase 激活、死亡诱导配体（包括 Fas 配体和 TRAIL）的表达，以及试图破坏宿主反应的病毒适应（例如病毒编码的凋亡抑制剂）例如，EBV 编码的 BALF1、KSHV 编码的 vFLIP 等）。实验性消除病毒编码的凋亡抑制剂可增强宿主细胞对病毒感染细胞的清除，并抑制病毒持续存在 - 例如：BALF1 可防止 EBV 持续存在，抑制 LAT 会抑制 HSV1 持续存在，并破坏 E6。或 E7 抑制 HPV 持续存在，可通过 IFI-16 检测累积的逆转录物来检测人类细胞中的 HIV 感染。 caspase 1 依赖性细胞焦亡，或整合酶介导的宿主 DNA“切口”的 DNA-PK 感应导致 p53 依赖性细胞凋亡。然而，这些感应机制并不能根除所有 HIV 感染的细胞，因此，HIV 不会持续存在。编码一种蛋白质，可以拮抗宿主根除受感染细胞的能力，但越来越多的证据表明，HIV 感染细胞比相应的未感染细胞更能抵抗细胞死亡，这是通过一种尚未明确的机制 TNF相关凋亡诱导因子（TRAIL）是一种分子，其主要功能是作为免疫监视的效应物，并且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，有大量数据。表明 TRAIL 在体内 HIV 感染过程中失调，并且有充分的证据表明，对 ART 细胞进行治疗可抑制外源性 TRAIL 感染 HIV 的患者，减少潜伏感染细胞的数量，如下所示：目前的研究提案涉及一种新型 TRAIL 剪接变体，我们已定量发现并命名为 TRAILshort，它是在体外和体内 HIV 感染过程中产生的。 (TRAILR2)，但不传递细胞死亡信号，而 TRAIL 与 TRAILR2 结合迅速导致细胞凋亡。此外，TRAILshort 的表达会阻止 TRAIL 参与细胞死亡。 TRAILR2，证明 TRAILshort 作为 TRAIL 介导的杀伤的显性负性抑制剂，因为 NK 细胞和 CD8+ CTL 通过 TRAIL（除了颗粒酶 B 等其他机制）杀死靶细胞，因此 TRAILshort 表达 NK 细胞和 CD8 T。我们的基本假设是，HIV 感染期间 TRAILshort 的产生可阻止 TRAIL 依赖性 HIV 清除机制，从而使 HIV 感染细胞得以持续存在。在该应用中，shRNA 介导的 TRAILshort 敲低不会改变未感染的 T 细胞的存活，而是选择性地增强对 HIV 感染细胞的杀伤，从而将 HIV 复制减少 3.5 个对数，并减少含有 HIV DNA 的细胞数量。我们的工作是充分了解和操纵 TRAILshort 的产生，以便正常的免疫机制有助于清除病毒感染的细胞，从而提供一种有助于治愈 HIV 的额外工具。这将通过以下方式进行充分探索： (i) 研究 TRAILshort 的调节，以确定抑制其产生的方法 (ii) 优化 HIV 感染患者的原代 CD4 T 细胞中 TRAILshort 的慢病毒敲低，并测试 TRAILshort 敲低是否会重新激活 HIV。与自体 CD8 T 细胞或 NK 细胞共培养，可减少离体 HIV 储存库大小 (iii) 评估 HIV 精英控制者、部分控制者和非控制者中的 TRAILshort 表达，并确定关联 TRAILshort 表达与 HIV 储存库大小有关。