NEF-CD4 PHYSICAL INTERACTION SENSED WITH THE YEAST 2-HYBRID SYSTEM

HIV-1 Nef protein has been known to induce downmodulation of CD4 recep tor. In order to test whether the two proteins physically interact, th e yeast two-hybrid system was exploited. A Saccharomyces cerevisiae st rain carrying a GAL4-responsive lacZ fusion gene was cotransformed wit h plasmids in which the Nef and the CD4 cytoplasmic domain (CD4cd) cod ing sequences were fused to either the DNA binding (DB) or the activat ion (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 numbe r of controls. Reporter activation was similarly observed when DB-Nef was cotransfected with the fusion A-CD4cd(aa 1-23). On the contrary, t he combination DB-Nef + A-CD4cd(aa 24-40) was inactive. Also, mutating the CD4cd Leu(20)-Leu(21) motif (known to be essential for both physi ological and Nef-induced CD4 endocytosis) to Ala(20)-Ala(21) abolished the GAL4 activity of DB-Nef + A-CD4cd. None of six DB-Nef derivatives in which Nef was partially deleted activated specifically the reporte r 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 particu lar, the Leu(20)-Leu(21) motif is essential. One can infer from these data that: (i) Nef-induced CD4 downmodulation involves a direct CD4-Ne f contact and (ii) CD4cd Leu(20)-Leu(21) is required in Nef-induced do wnmodulation, not simply as an endocytosis signal, but also as an esse ntial component of the Nef-binding moiety. (C) 1996 Academic Press, In c.