Liu L X, Margottin F, Le Gall S, Schwartz O, Selig L, Benarous R, Benichou S
Institut Cochin de Génétique Moléculaire, INSERM U332, Université Paris V, 24 Rue du Faubourg Saint-Jacques, 75014 Paris, France.
J Biol Chem. 1997 May 23;272(21):13779-85. doi: 10.1074/jbc.272.21.13779.
Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.
Nef是一种27 kDa的肉豆蔻酰化蛋白，在灵长类慢病毒中保守存在。在体内，猕猴体内的猿猴免疫缺陷病毒Nef对于产生高病毒载量和充分的病理效应是必需的。Nef在体外至少有三种主要作用，即诱导CD4下调、改变T细胞活化途径以及增强病毒感染性。我们利用酵母双杂交系统来鉴定与HIV-1 Lai Nef相互作用并可能介导Nef功能的细胞蛋白。分离出了一个编码新型硫酯酶的人类cDNA，硫酯酶是一种能切割硫酯键的酶。这种新型硫酯酶不同于先前克隆的I型和II型动物硫酯酶，但与大肠杆菌硫酯酶II同源。Nef与这种硫酯酶在体外相互作用，并在表达Nef的CEM细胞中被抗Nef抗体共免疫沉淀。来自无法下调CD4的人类免疫缺陷病毒-1（HIV-1）分离株的Nef等位基因不与硫酯酶反应或反应较弱。一个因缺乏与硫酯酶相互作用而被筛选出的HIV-1 Nef Lai突变体也无法下调CD4细胞表面表达。这些观察结果表明，这种人类硫酯酶是Nef诱导的CD4下调的细胞介质。
