Mechanisms of highly efficient HIV transfer at virological synapses

病毒突触高效 HIV 转移机制

基本信息

批准号：
7758258
负责人：
BENJAMIN K CHEN
金额：
$ 41.02万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7758258
关键词：
AIDS/HIV problem Actins Adhesives Affect Antibodies Antibody Formation Biological Assay Blocking Antibodies CCR5 gene CD4 Antigens CD4 Positive T Lymphocytes CXCR4 gene Cell Adhesion Cell Adhesion Molecules Cell Density Cell surface Cells Cellular biology Complement Critiques Cytoplasmic Tail DNA Sequence Rearrangement Data Endocytosis Ensure Epitopes Event Exposure to Fab Immunoglobulins Face Flow Cytometry Fluorescence Flushing Gagging Genetic HIV HIV Infections HIV-1 Helper-Inducer T-Lymphocyte Hour Image Immune Sera Infection Integrins Learning Life Maintenance Measures Mediating Membrane Fusion Molecular Cloning Monitor Mutation Nature North America Nucleic Acids Pathway interactions Patients Pharmaceutical Preparations Principal Investigator Process Reagent Research Research Personnel Resistance Role Signal Transduction Site Source Structural Protein Structure Suggestion Supporting Cell Surface Synapses T-Lymphocyte Testing Therapeutic Tissues Trypsin Vaccines Vesicle Viral Viral Pathogenesis Viral Proteins Virion Virus Virus Diseases Work cell motility chemotherapy crosslink improved in vivo inhibitor/antagonist interest microbicide mutant neutralizing monoclonal antibodies novel novel strategies pandemic disease particle polarized cell polyclonal antibody programs public health relevance research study response synaptogenesis transmission process uptake vaccine development virological synapse

项目摘要

DESCRIPTION (provided by applicant): The objective of our research is to reveal the mechanisms underlying T cell-to-T cell transfer of HIV. This enigmatic means of viral spread may be central to our understanding HIV transmission and viral dissemination within the host. Recent studies indicate that adhesive contacts between infected and uninfected T cells, called virological synapses (VS), mediate a highly efficient mode of infection. VS are intercellular adhesive structures that are driven by Env engagement, cell signaling, actin rearrangements and recruitment of cell adhesion molecules. Despite anecdotal evidence supporting this mode of viral spread, studies have yet to rigorously examine how VS transmission fundamentally differs from cell-free infection. Given the high density of cells in the tissue sites, its role is likely to be central to the establishment and maintenance of HIV infection. A major impasse to the study of cell-to-cell transfer has been the absence of quantitative assays to assess the efficiency of cell-mediated infection. To study transmission of HIV at the VS, we have created a novel, fluorescent molecular clone of HIV, called HIV Gag-iGFP. Infection with the virus renders both the infected cells and the infectious particles highly fluorescent, allowing us to track viral assembly and transmission with extraordinary sensitivity. Using flow cytometry we estimate that VS-mediated viral transfer is 18,000-fold more efficient than uptake of cell-free virus. In contrast to cell-free exposure, VS-transferred virus is rapidly internalized into trypsin-resistant compartments. VS-mediated transfer requires Env-CD4 receptor interactions, but is not blocked by viral membrane fusion inhibitors or by patient-derived neutralizing antisera capable of blocking cell-free virus. This resistance to neutralization by patient antisera is dependent upon an intact cytoplasmic tail of Env. Quantitative live imaging of the VS reveals that HIV-expressing cells are polarized and make stable, Env-dependent contacts with target cells through uropod-like structures. With spinning disk confocal imaging we can track the recruitment of viral proteins to the synapse in producer cells and the movement of virus-containing vesicles while they bud into target T cells. In this proposal, we test the hypothesis that Env on the surface of infected cells is involved in cell signaling events that trigger T cell adhesion, activating viral assembly and transmission from cell to cell through a vesicular compartment. Understanding the cell biology of cell-cell spread will be essential to learning how to block these processes in vivo. We will therefore reveal how cell-surface Env triggers the coordinated assembly and transfer into HIV-naive T cells. The work has significance for chemotherapy, microbicide and vaccine development against HIV. PUBLIC HEALTH RELEVANCE: The HIV/AIDS pandemic affects over 40 million worldwide and over 1.2 million people in North America. HIV primarily replicates in CD4 helper T cells and can induce adhesive infection-promoting intercellular structures between these cells, which are called virological synapses (VS). A better understanding of VS-mediated viral spread, will allow us to devise novel strategies to inhibit HIV spread with new drugs, microbicides or vaccines.

描述（由申请人提供）：我们研究的目的是揭示 HIV T 细胞间转移的机制。这种神秘的病毒传播方式可能对于我们理解艾滋病毒传播和病毒在宿主体内的传播至关重要。最近的研究表明，被感染和未感染的 T 细胞之间的粘附接触（称为病毒突触 (VS)）介导了一种高效的感染模式。 VS 是细胞间粘附结构，由 Env 接合、细胞信号传导、肌动蛋白重排和细胞粘附分子募集驱动。尽管有轶事证据支持这种病毒传播模式，但研究尚未严格检验 VS 传播与无细胞感染有何根本区别。鉴于组织部位细胞密度高，其作用可能对于艾滋病毒感染的建立和维持至关重要。细胞间转移研究的一个主要障碍是缺乏评估细胞介导的感染效率的定量分析。为了研究 HIV 在 VS 的传播，我们创建了一种新型 HIV 荧光分子克隆，称为 HIV Gag-iGFP。病毒感染使受感染的细胞和感染性颗粒都发出高度荧光，使我们能够以非凡的灵敏度追踪病毒的组装和传播。使用流式细胞术，我们估计 VS 介导的病毒转移效率比吸收无细胞病毒高 18,000 倍。与无细胞暴露相比，VS 转移的病毒迅速内化到胰蛋白酶抗性区室中。 VS介导的转移需要Env-CD4受体相互作用，但不能被病毒膜融合抑制剂或能够阻断无细胞病毒的患者来源的中和抗血清阻断。这种对患者抗血清中和的抵抗力依赖于 Env 的完整细胞质尾部。 VS 的定量实时成像显示，表达 HIV 的细胞是极化的，并通过尾足样结构与靶细胞建立稳定的、依赖于 Env 的接触。通过转盘共聚焦成像，我们可以追踪病毒蛋白向生产细胞突触的募集情况，以及含病毒囊泡在出芽进入目标 T 细胞时的运动。在本提案中，我们测试了这样的假设：受感染细胞表面的 Env 参与触发 T 细胞粘附、激活病毒组装以及通过囊泡在细胞之间传播的细胞信号转导事件。了解细胞间传播的细胞生物学对于学习如何在体内阻断这些过程至关重要。因此，我们将揭示细胞表面 Env 如何触发协调组装并转移到 HIV 初始 T 细胞中。这项工作对于针对艾滋病毒的化疗、杀菌剂和疫苗开发具有重要意义。公共卫生相关性：艾滋病毒/艾滋病的流行影响了全球超过 4000 万人和北美超过 120 万人。 HIV 主要在 CD4 辅助 T 细胞中复制，并可在这些细胞之间诱导粘附感染促进细胞间结构，称为病毒突触 (VS)。更好地了解 VS 介导的病毒传播，将使我们能够制定新策略，用新药物、杀菌剂或疫苗抑制 HIV 传播。