Geyer Matthias, Yu Haifeng, Mandic Robert, Linnemann Thomas, Zheng Yong-Hui, Fackler Oliver T, Peterlin B Matija
Department of Medicine, University of California, San Francisco, California 94143-0703, USA.
J Biol Chem. 2002 Aug 9;277(32):28521-9. doi: 10.1074/jbc.M200522200. Epub 2002 May 24.
Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.
Nef是人类和猿猴免疫缺陷病毒（HIV和SIV）的一种辅助蛋白，是病毒有效感染性和致病性所必需的。它会降低被感染细胞表面CD4的表达。V1H是液泡膜ATP酶（V-ATP酶）的调节亚基H。此前已发现Nef与V1H之间的相互作用有助于CD4的内化，这表明V1H可能将Nef与内吞机制联系起来。在本研究中，我们证明V1H在体外和体内均能与HIV-1的Nef中的C末端柔性环以及衔接蛋白复合物2（AP-2）的中链（μ2）结合。V1H与μ2的相互作用位点被定位到V1H中从第133位到363位的中央区域，该区域包含4个犰狳重复序列，以及μ2中从第1位到145位的N末端衔接蛋白结合结构域。将Nef与V1H融合可重现Nef的适当运输。即使在Nef不存在C末端柔性环的情况下，这种嵌合体也能使CD4内化。最后，阻断V1H的表达会降低Nef对病毒体感染性的增强作用。因此，V1H可作为Nef与AP-2之间相互作用的衔接蛋白发挥作用。
