HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion
HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion
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DOI：
10.1016/j.virol.2004.07.015
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发表时间：
2004-10-10
期刊：
影响因子：
3.7
通讯作者：
Greene, WC
中科院分区：
文献类型：
--
作者：
Cavrois, M;Neidleman, J;Greene, WC
我们最近描述了一种灵敏和特异的检测方法，可以检测HIV-1病毒粒子与广泛的靶细胞（包括原代CD 4细胞）的融合。该试验涉及使用含有β-内酰胺酶-Vpr（BlaM-Vpr）的病毒体，并使用β-内酰胺酶的荧光底物CCF 2加载靶细胞。由于Vpr与病毒核心强烈相关，因此BlaM-Vpr对CCF 2的有效切割可能需要病毒颗粒的脱壳。在这里，我们表明，成熟的病毒核心内的BlaM-Vpr有效地切割CCF 2，表明该测定法测量病毒体融合独立的脱壳。我们还表明，野生型和Nef缺陷型HIV-1病毒粒子融合与HeLa-CD 4细胞，SupT 1 T细胞和原代CD 4 T细胞的效率相当。由于Nef增强了病毒核心的细胞质递送并增加了病毒感染性，这些发现表明Nef增强了病毒进入的多步骤过程中的早期融合后事件。Nef作用的可能位点包括融合孔的扩大、病毒颗粒的增强的脱壳以及病毒核心通过位于质膜正下方的致密皮质肌动蛋白网络的更有效的通道。(C)2004年爱思唯尔公司All rights reserved.
We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane. (C) 2004 Elsevier Inc. All rights reserved.