Inhibition of cellular functions of HIV-1 Nef by artificial SH3 domains

SH3 domains regulate many normal and pathological cellular processes by gui ding specific protein interactions. Studies on binding of HIV-1 Nef to the SH3 domain of the Hck tyrosine kinase have indicated an important role for the SH3 RT-loop region in ligand binding. Here we have tested the potential of artificial Hck-derived SH3 domains carrying tailored RT-loops providing high affinity for Nef as intracellular inhibitors of Nef. These artificial SH3 domains efficiently associated with Nef in cells and thereby potently inhibited SH3-dependent Nef functions, such as association with p21-activat ed kinase-2 and induction of the transcription factor NFAT. On the other ha nd, biochemical and functional data indicated that the Nef-targeted SH3 dom ains were not prone to compete with normal SH3-mediated processes. Thus, RT -loop-modified SH3 domains represent a novel approach for selectively inter fering with cellular signaling events, which could be exploited in research as well as in therapeutic applications. (C) 2001 Academic Press.