Cavrois Marielle, Neidleman Jason, Yonemoto Wes, Fenard David, Greene Warner C
Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141-9100, United States.
Virology. 2004 Oct 10;328(1):36-44. doi: 10.1016/j.virol.2004.07.015.
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.
我们最近描述了一种灵敏且特异的检测方法，可检测HIV-1病毒粒子与多种靶细胞（包括原代CD4细胞）的融合。该检测方法涉及使用含有β-内酰胺酶-Vpr（BlaM-Vpr）的病毒粒子，并使靶细胞加载CCF2（一种β-内酰胺酶的荧光底物）。由于Vpr与病毒核心紧密结合，可能需要病毒粒子脱壳才能使BlaM-Vpr有效切割CCF2。在此，我们表明成熟病毒核心内的BlaM-Vpr可有效切割CCF2，这表明该检测方法测量的病毒粒子融合与脱壳无关。我们还表明野生型和Nef缺陷型HIV-1病毒粒子与HeLa-CD4细胞、SupT1 T细胞和原代CD4 T细胞融合的效率相当。由于Nef可增强病毒核心向细胞质的递送并增加病毒感染性，这些发现表明Nef在病毒进入的多步骤过程中增强了融合后早期事件。Nef作用的可能位点包括融合孔扩大、病毒粒子脱壳增强以及病毒核心更有效地穿过位于质膜下方的致密皮质肌动蛋白网络。
