Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.
Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.
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DOI：
10.1371/journal.ppat.1005709
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发表时间：
2016-06
期刊：
影响因子：
6.7
通讯作者：
Melikyan GB
中科院分区：
文献类型：
--
作者：
Francis AC;Marin M;Shi J;Aiken C;Melikyan GB
在靶细胞中消除锥形HIV-1衣壳是建立终身感染的先决条件。HIV-1进入的这一步，称为脱膜，是关键的，但人们对它的了解很少。在这里，我们报告了一种新的策略，可视化HIV-1的脱壳使用荧光标记的寡聚体形式的衣壳结合宿主蛋白亲环素A（CypA-DsRed），这是专门包装成病毒粒子通过高亲和力结合衣壳（CA）。单病毒成像显示，CypA-DsRed在透化/去除病毒膜后仍然与核心结合，并且CypA-DsRed和CA在体外和活细胞中从核心伴随丢失。损失率由核心稳定性调节，并在逆转录启动时加速。我们发现，大多数单核细胞在病毒融合后不久就失去了CypA-DsRed，而一小部分在几个小时内保持完整。感染后晚期的单颗粒追踪揭示了依赖于逆转录的CypA-DsRed的逐渐丧失。去包被发生在细胞质和核膜上。因此，我们的新型成像检测能够实现活细胞中单个HIV-1未涂层的时间分辨可视化，并揭示了这种不完全理解过程的先前未被认识的时空特征。HIV-1基因组和建立生产性感染所需的关键酶被包裹在由衣壳蛋白（CA）制成的锥形外壳中。在被释放到靶细胞的胞质溶胶中之后，锥形核心复合物经历一系列精心安排的步骤，包括脱壳（CA的损失）。HIV-1的脱壳仍然知之甚少，部分原因是缺乏直接的检测方法，使活细胞中的这一过程的研究。在这里，我们介绍了一种新的策略标记的HIV-1衣壳没有遗传修饰的CA蛋白。我们设计了一种新的荧光亲环蛋白A构建体，其以极高的亲合力结合衣壳，并且（1）有效地掺入病毒体而不损害感染性;（2）在病毒融合后保持与核心结合;以及（3）与CA一起从融合后核心中丢失。新的成像分析提供了新的见解的动力学和空间分布的HIV-1在活细胞中的脱壳。
Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. HIV-1 genome and key enzymes required for establishing productive infection are encased in a cone-shaped shell made of the capsid protein (CA). After being released into the cytosol of target cells, the cone-shaped core complex undergoes a series of carefully orchestrated steps, including uncoating (loss of CA). HIV-1 uncoating remains poorly understood, due in part to the lack of direct assays enabling studies of this process in living cells. Here, we introduce a novel strategy for labeling the HIV-1 capsid without genetically modifying the CA protein. We designed a novel fluorescent cyclophilin A construct that binds the capsid with an extremely high avidity and (1) efficiently incorporates into virions without compromising infectivity; (2) remains bound to cores after viral fusion; and (3) is lost from post-fusion cores along with CA. The novel imaging assay provides new insights into the kinetics and spatial distribution of HIV-1 uncoating in living cells.