Mechanisms of highly efficient HIV transfer at virological synapses

描述(由申请人提供)：我们研究的目的是揭示HIV的T细胞到T细胞转移的潜在机制。这种神秘的病毒传播方式可能是我们了解艾滋病毒传播和病毒在宿主内传播的核心。最近的研究表明，感染和未感染的T细胞之间的粘连接触，称为病毒学突触(VS)，介导了一种高效的感染模式。VS是由Env参与、细胞信号转导、肌动蛋白重排和细胞黏附分子募集所驱动的细胞间黏附结构。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不同。鉴于组织部位的细胞密度很高，它的作用很可能是建立和维持艾滋病毒感染的核心。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的感染效率。为了研究HIV在VS的传播，我们创造了一种新型的HIV荧光分子克隆，称为HIV Gag-iGFP。感染病毒使受感染的细胞和感染颗粒都具有高度的荧光，使我们能够以非凡的敏感性跟踪病毒的组装和传播。使用流式细胞术，我们估计VS介导的病毒转移的效率是摄取无细胞病毒的18,000倍。与无细胞暴露不同的是，VS转移的病毒会迅速内化到抵抗胰酶的隔膜中。VS介导的转移需要Env-CD4受体的相互作用，但不能被病毒膜融合抑制剂或患者来源的能够阻断无细胞病毒的中和抗血清所阻断。这种对患者抗血清中和的抵抗力依赖于Env的完整细胞质尾巴。VS的定量实时成像显示，表达HIV的细胞被极化，并通过尾足类结构与靶细胞进行稳定的、依赖于环境的接触。通过旋转圆盘共聚焦成像，我们可以跟踪病毒蛋白在产生细胞的突触中的募集情况，以及当病毒囊泡萌芽进入目标T细胞时的运动情况。在这个提议中，我们测试了这样的假设，即感染细胞表面的Env参与了触发T细胞黏附的细胞信号事件，激活了病毒的组装，并通过一个囊泡室在细胞之间传播。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是至关重要的。因此，我们将揭示细胞表面环境如何触发协同组装并转移到HIV-naive T细胞。这项工作对化疗、杀微生物剂和抗HIV疫苗的开发具有重要意义。 与公共卫生相关：艾滋病毒/艾滋病大流行影响到全世界4000多万人和北美120多万人。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性感染促进细胞间结构，称为病毒学突触(VS)。更好地了解VS介导的病毒传播，将使我们能够设计出新的策略，通过新药、杀微生物剂或疫苗来抑制艾滋病毒的传播。

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.