Rossi F, Gallina A, Milanesi G
Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Pavia, Italy.
Virology. 1996 Mar 1;217(1):397-403. doi: 10.1006/viro.1996.0130.
HIV-1 Nef protein has been known to induce downmodulation of CD4 receptor. In order to test whether the two proteins physically interact, the yeast two-hybrid system was exploited. A Saccharomyces cerevisiae strain carrying a GAL4-responsive lacZ fusion gene was cotransformed with plasmids in which the Nef and the CD4 cytoplasmic domain (CD4cd) coding sequences were fused to either the DNA binding (DB) or the activation (A) moiety of the GAL4 transcriptional activator. Both the DB-Nef + A-CD4cd and the DB-CD4cd + A-Nef combinations activated the reporter gene, weakly but specifically, as inferred by comparison with a number of controls. Reporter activation was similary observed when DB-Nef was cotransfected with the fusion A-CD4cd(aa 1-23). On the contrary, the combination DB-Nef + A-CD4cd(aa 24-40) was inactive. Also, mutating the CD4cd Leu20-Leu21 motif (known to be essential for both physiological and Nef-induced CD4 endocytosis) to Ala20-Ala21 abolished the GAL4 activity of DB-Nef + A-CD4cd. None of six DB-Nef derivatives in which Nef was partially deleted activated specifically the reporter when coexpressed with A-CD4cd. These findings suggest that CD4cd and Nef directly interact and that a largely complete Nef is required for the interaction. CD4cd aa 1-23 are sufficient for binding; in particular, the Leu20-Leu21 motif is essential. One can infer from these data that: (i) Nef-induced CD4 downmodulation involves a direct CD4-Nef contact and (ii) CD4cd Leu20-Leu21 is required in Nef-induced downmodulation, not simply as an endocytosis signal, but also as an essential component of the Nef-binding moiety.
已知HIV-1 Nef蛋白可诱导CD4受体下调。为了检测这两种蛋白是否发生物理相互作用，采用了酵母双杂交系统。将携带GAL4应答性lacZ融合基因的酿酒酵母菌株与质粒共转化，其中Nef和CD4胞质结构域（CD4cd）编码序列分别与GAL4转录激活因子的DNA结合（DB）或激活（A）部分融合。与多个对照相比推断，DB-Nef + A-CD4cd和DB-CD4cd + A-Nef组合均能微弱但特异性地激活报告基因。当DB-Nef与融合体A-CD4cd（第1至23位氨基酸）共转染时，同样观察到报告基因激活。相反，DB-Nef + A-CD4cd（第24至40位氨基酸）组合无活性。此外，将CD4cd的Leu20-Leu21基序（已知对生理性和Nef诱导的CD4内吞作用均至关重要）突变为Ala20-Ala21消除了DB-Nef + A-CD4cd的GAL4活性。当与A-CD4cd共表达时，六个Nef部分缺失的DB-Nef衍生物均未特异性激活报告基因。这些发现表明，CD4cd和Nef直接相互作用，且相互作用需要基本完整的Nef。CD4cd的第1至23位氨基酸足以结合；特别是Leu20-Leu21基序至关重要。从这些数据可以推断：（i）Nef诱导的CD4下调涉及CD4与Nef直接接触；（ii）CD4cd的Leu20-Leu21在Nef诱导的下调中是必需的，不仅作为内吞信号，而且作为Nef结合部分的重要组成部分。
