Mechanisms of highly efficient HIV transfer at virological synapses

描述（由申请人提供）：我们的研究目的是揭示HIV的T细胞到T细胞转移的机制。这种神秘的病毒传播方式可能是我们理解HIV传播和病毒在宿主内传播的核心。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，称为病毒学突触（VS），介导了一种高效的感染模式。VS是由Env结合、细胞信号、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。尽管坊间证据支持这种病毒传播模式，但研究尚未严格检查VS传播与无细胞感染有何根本区别。考虑到组织部位的高密度细胞，它的作用可能是建立和维持HIV感染的核心。细胞间转移研究的一个主要僵局是缺乏定量分析来评估细胞介导感染的效率。为了研究艾滋病毒在VS中的传播，我们创造了一种新的，荧光分子克隆的艾滋病毒，称为HIV Gag-iGFP。病毒感染使受感染的细胞和感染颗粒都具有高度荧光，使我们能够以非凡的灵敏度跟踪病毒的组装和传播。利用流式细胞术，我们估计vs介导的病毒转移比无细胞病毒的吸收效率高18,000倍。与无细胞暴露相反，vs转移的病毒迅速内化到抗胰蛋白酶的隔室中。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性抗血清阻断。这种对患者抗血清中和的抵抗依赖于Env完整的细胞质尾部。VS的定量实时成像显示，表达hiv的细胞是极化的，并通过类似于尾足的结构与靶细胞进行稳定的、依赖于环境的接触。通过旋转盘共聚焦成像，我们可以追踪病毒蛋白在产生细胞突触上的募集，以及含病毒囊泡在进入靶T细胞时的运动。在本提案中，我们验证了感染细胞表面的Env参与触发T细胞粘附的细胞信号事件，激活病毒组装并通过囊泡室在细胞间传播的假设。了解细胞-细胞扩散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-naive T细胞中。这项工作对艾滋病的化疗、杀微生物剂和疫苗的开发具有重要意义。

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.