Does HIV Enter Doomed Cells

描述（由申请方提供）：在最佳体外条件下，HIV生命周期需要约24小时才能完成。因此，不可逆地进入已经启动凋亡程序的细胞是HIV的死胡同，因为这些细胞通常在24小时内死亡。在体内，它们可以通过吞噬细胞识别表面标记物（如外翻PS）而更快地清除。未能与凋亡细胞不可逆融合的病毒粒子将比那些进展超过包膜结合以融合的病毒粒子具有选择性优势。这种区分的潜在重要性由于HIV需要活化的T细胞来有效进入、逆转录和整合的事实而增加。由于>90%的生理活化的T细胞在约5天的时间内注定进行活化诱导的细胞死亡（AICD），因此HIV将有合理的机会遇到活化的凋亡的CD 4+淋巴细胞或水泡。这在肠道超急性疾病期间尤其如此，其中在显著AICD的情况下存在大量CD 4 + T细胞感染。在超急性期缺乏特异性抗病毒免疫反应的情况下，避免凋亡细胞进入可能是一种主要的选择压力。 我们假设这种压力导致了HIV避免与凋亡细胞不可逆融合的能力，并且如果结合则保持感染性。我们进一步假设HIV+ DC不会通过感染性突触将HIV不可逆地转移到凋亡细胞。我们还将测试两个不同但相关的假设：凋亡细胞对HIV包膜脱落源的趋化性有缺陷，非特异性结合病毒粒子的内吞有缺陷。我们的具体目标是1）使用R5包膜、病毒体和HIV+ DC严格测试这些假设，和2）检查受体共加帽的损失和LFA-1的损失，作为HIV可以感知和避免凋亡的潜在宿主细胞的潜在机制。我们的长期目标是利用HIV的这一特征来设计预防和治疗方案，使病毒避免进入健康细胞，这些细胞在HIV感染的一个或多个关键方面模拟凋亡细胞。 我们的方法将使用生理相关信号来触发CD 4 + T细胞中的AICD，并在随后的时间点将其暴露于HIV或HIV+ DC。通过荧光染料检测线粒体膜的变化，对细胞进行早期凋亡分选，并通过双重荧光病毒体进入、BlaM-vpr活性和细胞内基因组病毒RNA的定量RT-PCR，结合HIV从细胞表面的蛋白水解剥离，检测HIV的表面结合与不可逆进入。将通过挽救培养物评估保留在凋亡细胞表面上的HIV的免疫活性。这个想法是完全新颖的，并且非常重要，因为它将提供对逆转录病毒包膜的进化和结构的洞察，并且将是第一次证明在感染之前由病原体对宿主细胞适应性的外部探测。另外的意义来自于利用我们的发现进行预防和治疗的潜力，通过对关键细胞成分赋予短暂的模拟骨化症的表型。 公共卫生相关性：艾滋病毒是一种适应性很强的病毒，它在单个感染者体内竞争性地进化，以避免被宿主防御或药物消除，并确保在体内复制的最佳位点。在数十亿个病毒粒子之间的这种达尔文主义竞赛中，复制和存活方面的优势迅速导致选择负责任的性状，例如病毒避免进入已经启动自我毁灭调节程序的注定失败的潜在宿主细胞或无法有效复制病毒的静止细胞的能力。我们的目标是证明这种回避机制，并最终希望暂时模拟细胞的关键方面，向HIV发出程序性细胞死亡（或静止）的信号，从而诱使病毒避开这些细胞。

DESCRIPTION (provided by applicant): The HIV life cycle requires ~ 24 hours for completion, under optimal in vitro conditions. Thus, irreversible entry into cells that have initiated an apoptotic program is a dead end for HIV, as these cells generally die within 24 hours. In vivo they may be cleared even more quickly by phagocytes recognizing surface markers such as everted PS. Virions that failed to irreversibly fuse with apoptotic cells would have a selective advantage over those that progressed beyond envelope binding to fusion. The potential importance of such discrimination is increased by the fact that HIV requires activated T cells for efficient entry, reverse transcription and integration. Since >90% of physiologically activated T cells are destined for activation induced cell death (AICD) over a period of ~ 5 days, there is a reasonable chance that HIV will encounter activated apoptotic CD4+ lymphocytes or blebs. This is particularly true during hyperacute disease in the gut, where there is massive CD4+ T cell infection in the setting of significant AICD. In the absence of specific anti-viral immune responses during the hyperacute phase, avoidance of apoptotic cell entry could be one dominant selective pressure. We hypothesize that this pressure has resulted in an ability of HIV to avoid irreversible fusion with apoptotic cells, and to remain infectious if bound. We further hypothesize that HIV+ DCs will not irreversibly transfer HIV to apoptotic cells via infectious synapses. We will also test two distinct but related hypotheses: apoptotic cells are defective in chemotaxis toward a source of shed HIV envelope, and defective in endocytosis of non-specifically bound virions. Our specific aims are to 1) rigorously test these hypotheses using R5 envelope, virions, and HIV+ DCs, and 2) examine loss of receptor co-capping, and loss of LFA-1, as potential mechanisms whereby HIV may sense and avoid apoptotic potential host cells. Our long term objective is to exploit this feature of HIV to design prophylactic and treatment options that trick the virus into avoiding entry into healthy cells that have been made to mimic apoptotic cells in one or more aspects crucial to HIV infection. Our methods will use physiologically relevant signals to trigger AICD in CD4+ T cells, and expose them to HIV, or HIV+ DCs, at subsequent time points. Cells will be sorted for early apoptosis by fluorescent dye detected changes in mitochondrial membranes, and surface binding vs. irreversible entry of HIV will be detected by dual fluorescent virion entry, BlaM-vpr activity, and quantitative RT-PCR of intracellular genomic viral RNA, in conjunction with proteolytic stripping of HIV from the cell surface. Infectivity of HIV retained on the surface of apoptotic cells will be assessed by rescue cultures. This idea is completely novel, and is highly significant because it would provide insight into the evolution and structure of retroviral envelope, and would be the first demonstration of external probing by a pathogen of host cell fitness, prior to infection. Additional significance derives from the potential to exploit our findings for prevention and treatment, by pharmacologically conferring on key cellular components a transient apoptosis-mimicking phenotype. PUBLIC HEALTH RELEVANCE: HIV is a highly adaptable virus that evolves competitively within a single infected person to avoid elimination by host defenses or drugs, and to secure the best sites for replication within the body. In this Darwinian race among billions of individual virions, advantages in replication and survival rapidly lead to selection of the responsible traits, such as the ability of a virus to avoid entering a doomed potential host cell that has initiated a regulatory program of self-destruction, or a quiescent cell that cannot replicate virus efficiently. We aim to demonstrate this avoidance mechanism, and, eventually, hope to pharmacologically mimic, transiently, key aspects of the cell that signal programmed cell death (or quiescence) to HIV, thereby tricking the virus into avoiding such cells.