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

 描述(申请人提供)：病原性病毒感染和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。检测病毒感染的宿主效应机制(例如，caspase激活，包括Fas配体和TRAIL在内的死亡诱导配体的表达)，以及试图颠覆宿主反应的病毒适应(例如，病毒编码的凋亡抑制物，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物可增强病毒感染细胞对宿主细胞的清除，并降低病毒持久性-例如：抑制BALF1可阻止EBV持久性，抑制LAT可抑制HSV1持久性，干扰E6或E7可抑制HPV持久性。HIV感染可以在人类细胞中检测到，例如通过IFI-16检测累积的逆转录导致caspase 1依赖的下垂，或者DNA-PK检测整合酶介导的宿主DNA“缺口”导致P53依赖的细胞凋亡。然而，这些感知机制并不能根除所有感染艾滋病毒的细胞，因此艾滋病毒是持久的。人类免疫缺陷病毒(HIV)编码的一种蛋白可以对抗宿主清除感染细胞的能力，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，其机制尚不明确，肿瘤坏死因子相关的凋亡诱导因子(TRAIL)是一种主要功能是免疫监视的分子，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，大量数据表明，TRAIL在体内感染HIV的过程中是失调的，有充分的证据表明，用外源TRAIL抑制HIV感染患者的细胞，减少了潜伏感染细胞的数量，通过定量共培养检测不到的复制能力病毒水平来衡量。目前的研究方案涉及一种新的TRAIL剪接变异体，我们已经发现并命名为TRAILShort，它是在体内和体外感染HIV时产生的。TRAILShort与TRAIL受体2(TRAILR2)结合，但不传递细胞死亡信号，而TRAIL与TRAILR2结合迅速导致细胞死亡。此外，TRAILShort的表达阻止了TRAILR2与TRAILR2的结合，表明TRAILShort是TRAIL介导的杀伤的显性负抑制因子。由于NK细胞和CD8CTL通过TRAIL(加上颗粒酶B等其他机制)杀伤靶细胞，因此TRAILShort的表达降低了NK细胞和CD8T细胞的杀伤活性。我们的基本假设是，HIV感染期间TRAIL的短期产生阻止了TRAIL依赖的HIV清除机制，从而使HIV感染细胞得以持续存在。这一假说得到了本申请中新的初步数据的支持，其中shRNA介导的TRAILShort的敲除不会改变未感染的T细胞的存活，但选择性地增强对HIV感染细胞的杀伤，从而将HIV复制减少3.5logs，并减少含有HIV DNA的细胞数量。我们工作的长期目标是充分了解和操纵TRAIL短链的产生，以便正常的免疫机制能够有助于清除病毒感染的细胞，从而提供一个有助于治愈艾滋病毒的额外工具。将通过以下方式充分探讨这一点：(I)研究TRAILShort的监管，以确定抑制其生产的方法。(Ii)优化慢病毒敲除HIV感染患者的原代CD4T细胞中TRAILShort的表达，并测试TRAILShort敲除后HIV的重新激活，加上或不与自体CD8 T细胞或NK细胞共同培养，是否可以减少体外HIV储存库的大小。(Iii)评估TRAILShort在HIV精英控制者、部分控制者和非控制者中的表达，并确定与 TRAIL短表达与HIV储存库大小有关。

 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.