Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the "R-clamp" - that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution. AUTHOR SUMMARYViral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.