Does HIV Enter Doomed Cells

描述(由申请人提供)：在最佳的体外条件下，艾滋病毒的生命周期需要~24小时才能完成。因此，不可逆转地进入启动了凋亡程序的细胞是艾滋病毒的死胡同，因为这些细胞通常在24小时内死亡。在体内，它们可能会被吞噬细胞识别外翻PS等表面标志而更快地清除。与那些超越包膜结合到融合的病毒粒子相比，未能与凋亡细胞不可逆转地融合的病毒粒子将具有选择性优势。艾滋病毒需要激活的T细胞才能有效进入、逆转录和整合，这一事实增加了这种歧视的潜在重要性。由于90%的生理激活的T细胞在~5天内被指定为激活诱导细胞死亡(AICD)，因此HIV有合理的机会遇到激活的凋亡的CD4+淋巴细胞或水泡。这在肠道的超急性疾病中尤其如此，在严重的AICD的情况下，有大量的CD4+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有能力避免与凋亡细胞不可逆转的融合，并在结合时保持传染性。我们进一步假设，HIV+DC不会通过感染性突触不可逆转地将HIV转移到凋亡细胞。我们还将测试两个不同但相关的假设：凋亡细胞对脱落的HIV包膜来源的趋化作用存在缺陷，对非特异性结合的病毒粒子的内吞存在缺陷。我们的具体目标是1)使用R5包膜、病毒粒子和HIV+DC严格测试这些假说，2)检查受体共帽的丢失和LFA-1的丢失，作为HIV可能感知和避免潜在宿主细胞凋亡的潜在机制。我们的长期目标是利用艾滋病毒的这一特征来设计预防和治疗方案，欺骗病毒避免进入健康细胞，这些细胞已经在一个或多个对艾滋病毒感染至关重要的方面模仿了凋亡细胞。我们的方法将使用生理上相关的信号来触发CD4+T细胞中的AICD，并在随后的时间点将它们暴露在HIV或HIV+DC中。将通过荧光染料检测线粒体膜的变化来分选细胞进行早期凋亡，并将通过双重荧光病毒粒子进入、BLAM-VPR活性和定量RT-PCR结合HIV从细胞表面的蛋白水解性剥离来检测HIV的表面结合和不可逆进入。保留在凋亡细胞表面的艾滋病毒的传染性将通过救援培养进行评估。这一想法是完全新颖的，具有非常重要的意义，因为它将提供对逆转录病毒包膜的进化和结构的洞察，并将首次证明病原体在感染前对宿主细胞适合性的外部探测。更重要的是，通过对关键细胞成分进行药理学研究，开发我们的发现用于预防和治疗的潜力，这是一种瞬时的类似细胞凋亡的表型。 与公共卫生相关：艾滋病毒是一种高度适应性的病毒，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置。在这个几十亿个体病毒粒子之间的达尔文种族中，复制和生存的优势迅速导致了对负责特征的选择，例如病毒避免进入注定要进入的潜在宿主细胞的能力，该宿主细胞已经启动了自我毁灭的调节程序，或者是一个静止的细胞，不能有效地复制病毒。我们的目标是证明这种回避机制，并最终希望在药物上暂时模拟细胞的关键方面，向艾滋病毒发出程序性细胞死亡(或静止)的信号，从而诱使病毒避开这样的细胞。

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.