Does HIV Enter Doomed Cells

说明（由申请人提供）：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，它们可能被吞噬细胞识别表面标记物（如everted PS）更快地清除。无法与凋亡细胞不可逆融合的病毒粒子比那些从包膜结合发展到融合的病毒粒子具有选择性优势。HIV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%的生理活化的T细胞在5天内注定会发生活化诱导细胞死亡（AICD），因此HIV有合理的机会遇到活化的凋亡CD4+淋巴细胞或泡。在肠超急性疾病中尤其如此，在严重AICD的情况下，存在大量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个主要的选择压力。我们假设这种压力导致HIV能够避免与凋亡细胞的不可逆融合，并在结合后保持传染性。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中。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包膜来源的趋化性缺陷，以及非特异性结合病毒粒子的内吞作用缺陷。我们的具体目标是1)使用R5包膜、病毒粒子和HIV+ dc严格测试这些假设，2)检查受体共盖层的丢失和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点，设计预防和治疗方案，使病毒避免进入健康细胞，这些细胞在艾滋病毒感染的一个或多个关键方面模仿凋亡细胞。我们的方法将使用生理相关信号触发CD4+ 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早期凋亡进行分类，通过双荧光病毒粒子进入、BlaM-vpr活性和细胞内基因组病毒RNA的定量RT-PCR检测表面结合与不可逆进入HIV，并结合从细胞表面蛋白水解剥离HIV。保留在凋亡细胞表面的HIV的传染性将通过抢救培养来评估。这个想法是完全新颖的，而且非常重要，因为它将提供对逆转录病毒包膜的进化和结构的深入了解，并且将首次证明病原体在感染之前对宿主细胞适应性进行外部探测。通过药理学赋予关键细胞成分一种短暂的细胞凋亡模拟表型，我们的研究结果有可能用于预防和治疗。

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.