Greenberg M E, Bronson S, Lock M, Neumann M, Pavlakis G N, Skowronski J
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
EMBO J. 1997 Dec 1;16(23):6964-76. doi: 10.1093/emboj/16.23.6964.
The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.
人类免疫缺陷病毒（HIV）和猿猴免疫缺陷病毒（SIV）的nef基因对艾滋病发病机制至关重要。其在体内的功能尚不清楚，但在体外，Nef的天然分离株通过加速细胞表面CD4分子（T细胞抗原受体和病毒受体的一个组成部分）的内吞作用来下调其表达。我们使用了由天然HIV-1 NA7 Nef与绿色荧光蛋白（GFP）的强荧光突变体融合而成的嵌合蛋白，以将Nef功能与人类CD4阳性Jurkat T细胞中的细胞内定位相关联。NA7-GFP融合蛋白与网格蛋白包被的成分共定位，包括网格蛋白和AP-2衔接蛋白复合物的β亚基，其在离散位置的分布与质膜上网格蛋白包被的正常细胞分布一致。NA7-GFP蛋白也存在于细胞的核周区域，这可能反映了高尔基体。来自CD4阴性成纤维细胞系的证据表明，NA7-GFP与网格蛋白包被成分的共定位不需要CD4分子的表达。对一大组含有突变Nef部分的嵌合分子的分析表明，N端膜靶向信号与Nef分子无序环中的其他元件协同作用，使Nef蛋白与AP-2衔接复合物在细胞边缘共定位。NA7-GFP的这种定位与表面CD4的下调相关，但并不充分，还需要Nef的至少一种其他功能。在共表达CD4和NA7-GFP的T细胞中，细胞表面的CD4重新分布成与NA7-GFP共定位的离散模式。我们的观察结果表明，NA7-GFP在质膜上与含AP-2的网格蛋白包被在物理上接近，这意味着Nef在该位置直接或间接与含AP-2包被的一个成分相互作用。这一证据支持了一个模型，即Nef通过质膜上含AP-2的网格蛋白包被将CD4招募到内吞机制中。
