Does HIV Enter Doomed Cells

HIV是否会进入注定失败的细胞

基本信息

批准号：
7843379
负责人：
SUJATHA IYENGAR
金额：
$ 26.42万
依托单位：
HACKENSACK UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7843379
关键词：
Address Anti-Retroviral Agents Apoptosis Apoptotic B-Lymphocytes Behavior Binding Biological Blast Cell Bulla CCR5 gene CD14 gene CD4 Positive T Lymphocytes CD8B1 gene CXCR4 gene Caliber Cell Death Cell membrane Cell surface Cells Chemotaxis Color Complement Receptor Confocal Microscopy Detection Discrimination Disease Dyes Endocytosis Evolution Excision Fluorescence Fluorescent Dyes Future Genomics Goals HIV HIV Envelope Protein gp120 HIV Infections Host Defense Hour Immune response Immunoglobulin M In Vitro Individual Infection Ingestion Integral Membrane Protein Knowledge Label Lactamase Lamivudine Lead Life Cycle Stages Lymphoid Tissue Macaca Membrane Microdomains Methods Modeling Persons Phagocytes Pharmaceutical Preparations Phase Phenotype Population Prevention Production Prophylactic treatment Publishing RNA Race Reaction Reading Recruitment Activity Residual state Reverse Transcriptase Polymerase Chain Reaction Reverse Transcription SIV Sampling Satellite Viruses Secure Serine Signal Transduction Site Sorting - Cell Movement Source Speed Staging Structure Surface Synapses System T-Lymphocyte Techniques Testing Time Treatment-Related Cancer Trypsin Viral Virion Virus Work Zidovudine annexin A5 apoptosis in lymphocytes base chemokine design fitness in vivo innovation insight interest lymphocyte proliferation macrophage mitochondrial membrane novel pathogen pressure programs public health relevance receptor research study response trait transmission process viral RNA

项目摘要

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.

描述（由申请人提供）：在最佳体外条件下，艾滋病毒生命周期需要约24小时才能完成。因此，由于这些细胞通常在24小时内死亡，因此不可逆转地进入已经启动凋亡程序的细胞是HIV的死胡同。在体内，可以通过识别表面标记（例如ps）的表面标记的吞噬细胞更快地清除它们。与凋亡细胞不可逆地融合的病毒体将比超越包膜与融合结合的侵蚀性具有选择性的优势。艾滋病毒需要激活的T细胞以有效进入，逆转录和整合，这种歧视的潜在重要性将增加。由于> 90％的生理活化的T细胞注定要在约5天内激活诱导细胞死亡（AICD），因此HIV很有可能遇到活化的凋亡CD4+淋巴细胞或BLEB。在肠道中急性疾病中尤其如此，在大量AICD的情况下，CD4+ T细胞感染大量。在超急性期没有特定的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。我们假设这种压力已导致HIV避免与凋亡细胞不可逆的融合的能力，并保持感染力。我们进一步假设HIV+ DC不会通过传染性突触不可逆地将HIV转移到凋亡细胞中。我们还将检验两个不同但相关的假设：凋亡细胞在趋化型HIV包膜来源的趋化性中有缺陷，并且在非特异性结合的病毒体的内吞作用中有缺陷。我们的具体目的是1）使用R5包膜，病毒体和HIV+ DC严格检验这些假设，以及2）检查受体共封码的丢失以及LFA-1的丢失，作为HIV可能感知并避免凋亡势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性和治疗方案，诱使病毒避免进入健康细胞，这些细胞已在一个或多个方面模仿对HIV感染至关重要的凋亡细胞。我们的方法将使用生理相关的信号来触发CD4+ T细胞中的AICD，并在随后的时间点将其暴露于HIV或HIV+ DCS。通过荧光染料检测到线粒体膜的变化，将对细胞进行分类，并将表面结合与HIV的不可逆进入，将通过双荧光病毒率进入，BLAM-VPR活性和定量的RT-PCR来检测HIV的细胞内基因组病毒RNA的定量RT-PCR。保留在凋亡细胞表面的HIV的感染力将通过救援培养物评估。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演化和结构的见解，并且是感染前宿主细胞适应性的病原体对外部探测的首次演示。额外的显着性源于利用我们的发现进行预防和治疗的潜力，从药理上赋予关键细胞成分的瞬态凋亡模仿表型。公共卫生相关性：艾滋病毒是一种高度适应性的病毒，在单个感染者中竞争性地发展，以避免被宿主防御或药物消除，并确保在体内复制的最佳地点。在数十亿个单个病毒体中的达尔文人种族中，复制和生存方面的优势迅速导致了负责任的特征，例如病毒避免进入注定的潜在宿主细胞的能力，该细胞已经启动了自我毁灭的调节计划，或者无法有效地重复病毒。我们的目的是证明这种回避机制，并最终希望在药理学上模仿，瞬时地模仿细胞的关键方面，该细胞向HIV发出了编程细胞死亡（或静止），从而诱使病毒避免了这种细胞。