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：
8016579
负责人：
BENJAMIN K CHEN
金额：
$ 40.61万
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/8016579
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 Cytoplasmic Tail DNA Sequence Rearrangement Data Endocytosis Ensure Epitopes Event Exposure to Fab Immunoglobulins 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 Monoclonal Antibodies 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艾滋病毒T细胞对T细胞转移的基础机制。这种神秘传播的神秘手段可能是我们理解宿主内的HIV传播和病毒传播的核心。最近的研究表明，被感染和未感染的T细胞（称为病毒学突触（VS））之间的粘合剂接触介导高效的感染方式。 V是由环境参与，细胞信号传导，肌动蛋白重排和细胞粘附分子募集驱动的细胞间粘附结构。尽管轶事证据支持这种病毒传播模式，但研究尚未严格研究与无细胞感染的基本上不同的传播。鉴于组织部位中细胞的密度高，其作用可能是HIV感染的建立和维持的核心。对细胞向细胞转移研究的主要僵局是没有定量测定来评估细胞介导的感染的效率。为了研究HIV在VS上的传播，我们创建了一种新型的荧光分子克隆HIV，称为HIV GAG-IGFP。感染该病毒会导致感染的细胞和传染性颗粒高度荧光，从而使我们能够以非常敏感性跟踪病毒组装和传播。使用流式细胞仪，我们估计VS介导的病毒转移比无细胞病毒的摄取效率高18,000倍。与无细胞的暴露相反，VS转移的病毒迅速内化为耐胰蛋白酶的隔室。 VS介导的转移需要ENV-CD4受体相互作用，但不会被病毒膜融合抑制剂或能够阻止无细胞病毒的患者衍生的中和抗血清所阻断。患者抗血清对中和的抗性取决于Env的完整细胞质尾巴。 VS的定量实时成像表明，表达艾滋病毒的细胞是极化的，并通过乌拉托德样结构与靶细胞与目标细胞建立了稳定，依赖于目标。通过旋转盘共聚焦成像，我们可以跟踪病毒蛋白在生产者细胞中的突触募集以及含病毒囊泡的运动，同时它们会芽到靶T细胞中。在此提案中，我们检验了一个假设，即感染细胞表面的ENV参与了触发T细胞粘附，激活病毒组装并通过囊泡室从细胞到细胞的传播的细胞信号事件。了解细胞细胞扩散的细胞生物学对于学习如何在体内阻止这些过程至关重要。因此，我们将揭示细胞表面ENV如何触发协调的组装并转移到HIV-NOIVE T细胞中。这项工作对于针对HIV的化学疗法，杀菌剂和疫苗发育具有重要意义。公共卫生相关性：艾滋病毒/艾滋病大流行在北美影响全球超过4000万人，超过120万人。 HIV主要在CD4辅助T细胞中复制，并可以在这些细胞之间诱导促进感染感染的细胞间结构，这些细胞称为病毒学突触（VS）。对VS介导的病毒传播的更好理解将使我们能够制定新的策略，以抑制新药，菌心或疫苗的艾滋病毒传播。