HIV-2 and SIV Nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution

The nef gene is required for optimal viral spread of human and simian immun odeficiency viruses. However, the molecular mechanisms underlying the actio n of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian imm unodeficiency viruses and SH3 domains from Src family kinases, Using the ye ast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SN and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Ne f structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that locali ze in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are sy stematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respec tively). We demonstrate that site-directed mutagenesis of these residues in SIVmac239 Nef suffices to restore Hck SH3 binding and co immunoprecipitati on with full-length Hck from transfected cells, Our findings identify funda mental mechanistic differences in targeting of Src family kinases by HN and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "poc ket" proximal to the canonical proline-rich motif may be a common feature f or SH3 recognition by their natural ligands.