HIV Fusion to CD4 T Cells and Dendritic Cells

描述（由申请人提供）：干扰病毒进入是一种很有前途的中断HIV-1生命周期的策略。理想情况下，HIV-1融合应该在最相关的生理条件下进行研究，包括使用实际的病毒粒子和原代细胞。我们开发了一种敏感和特异性的基于HIV-1病毒粒子的检测方法，允许研究含有（b -内酰胺酶vpr）的病毒粒子与装载有CCF2（一种荧光性b -内酰胺酶底物）的主要靶细胞的融合。我们假设HIV-1病毒粒子与生物学相关靶细胞融合的研究，包括原发性CD4 T淋巴细胞亚群和成熟或激活状态不同的树突状细胞（dc），将揭示在病毒发病过程中起主要作用的重要差异。在特异性目标1中，我们将分析人类扁桃体组织中HIV-1与原发性CD4 T淋巴细胞亚群的融合。我们的初步研究表明，HIV-1与记忆细胞的融合水平高于与初始CD4 T细胞的融合水平。同样，细胞的激活状态也会影响细胞对融合的敏感性。我们将研究这些差异是否反映了趋化因子辅助受体表达的变化或HIV-1结合的变化。在特异性目标2中，我们将研究DC成熟和HIV趋向性是否影响HIV-1病毒粒子与DC融合的动力学以及融合发生的室。我们已经观察到成熟和未成熟的树突状细胞对R5-和x4 -型HIV-1病毒粒子融合的敏感性存在显著差异。我们将描述融合动力学，并研究HIV-1病毒粒子从内体区室融合的可能性。与此同时，我们将继续探索广泛的体内dc对X4-和r5 -向HIV-1融合的易感性。在特异性目标3中，我们将比较通过粘膜上皮传播的HIV-1病毒粒子与未通过粘膜上皮传播的HIV-1病毒粒子的融合。对于这些研究，我们将采用基于病毒粒子的融合试验来研究原代分离物。这些病毒粒子将从hiv -1感染患者的供体-受体配对中扩增出来。将该方法应用于初级分离株的分析是一项技术飞跃，将对未来的研究有用。拟议的研究有望推进我们对HIV-1融合和控制这一关键病毒进入事件的细胞决定因素的理解。这些发现可能会提出新的策略来阻断HIV-1生命周期的早期阶段，并阻断HIV-1在体内的水平传播。

DESCRIPTION (provided by applicant): Interfering with viral entry is a promising strategy for interrupting the HIV-1 life cycle. Ideally, HIV-1 fusion should be studied under the most physiologically relevant conditions possible, including the use of actual virions and primary cells. We developed a sensitive and specific HIV-1 virion-based assay that permits the study of fusion of virions containing (B-lactamase-Vpr to primary target cells loaded with CCF2, a fluorogenic B-lactamase substrate. We hypothesize that the study of the fusion of HIV-1 virions to biologically relevant target cells, including subsets of primary CD4 T lymphocytes and dendritic cells (DCs) varying in their state of maturation or activation, will reveal important differences that centrally contribute to viral pathogencsis. In Specific Aim 1, we will analyze HIV-1 fusion to subsets of primary CD4 T lymphocytes in human tonsillar tissue. Our preliminary studies show higher levels of HIV-1 fusion to memory than to naive CD4 T cells. Similarly, the state of cellular activation could also influence the susceptibility of cells to fusion. We will investigate whether these differences reflect changes in chemokine coreceptor expression or changes in HIV-1 binding. In Specific Aim 2, we will investigate whether DC maturation and HIV tropism influence the kinetics of HIV-1 virion fusion to DCs and the compartment from which fusion occurs. We have observed dramatic differences in the susceptibility of mature and immature DCs to fusion of R5- and X4-tropic HIV-1 virions. We will characterize the fusion kinetics and investigate the possibility that HIV-1 virions fuse from endosomal compartments. In parallel, we will continue to explore the susceptibility of a wide range of in vivo DCs to X4- and R5-tropic HIV-1 fusion. In Specific Aim 3, we will compare fusion of HIV-1 virions that were transmitted across the mucosal epithelia versus those that were not transmitted. For these studies, we will adapt the virion-based fusion assay to study primary isolates. These virions will be amplified from donor-recipient pairs of HIV-1-infected patients. Adaptation of the assay for analysis of primary isolates is a technological leap forward that will be useful for future studies. The proposed studies promise to advance our understanding of HIV-1 fusion and the cellular determinants that govern this key viral entry event. The findings could suggest new strategies to block this early step in the HIV-1 life cycle and to interrupt the horizontal transmission of HIV-1 in vivo.