From What Is to What If : Unleashing the Power of Imagination to Create the Future We Want

The nef gene of HIV and simian immunodeficiency virus (SIV) encodes a ∼27-34 kDa myristylated protein that plays an important role in the progression of AIDS. Nef has multiple effects on virus replication that are regulated by an adaptor protein function that mediates diverse interactions with a variety of intracellular ligands. To gain a better understanding of the regulation of Nef, we investigated molecular determinants of Nef function. We first targeted the N-terminus of Nef and identified a novel serine/threonine kinase activity (termed the SNTK activity) that associates with SIVmac239 Nef, but not with HIV-lSF2 Nef. Intriguingly, amino acids required for SNTK activity were similar to those reported to mediate the myristyl switch of the Mason-Pfizer Monkey Virus matrix protein, suggesting that SIV Nef may possess an analogous function. We further explored the functional contribution of the central and C-terminal regions of Nef and mapped the p21-activated kinase (PAK)-activation domains of HIV and SIV Nef. Both PAK-activation domains were found to be highly conserved and contain amino acid sequences that form the structured globular core domain of Nef. Moreover, amino acid substitutions within the PAK-activation domain were found to also influence downregulation of CD4 and/or MHC I cell surface receptors as well the intracellular localization of Nef, with some substitutions having pleiotropic effects, suggesting that the core domain of Nef contains overlapping functional domains. Accordingly, conformations of Nef that mediate its function were identified by analyzing existing structures of HIV-1 Nef. We further investigated the regulation of Nef by its intracellular localization and observed that nuclear localization was induced by mutation of the myristylation signal of both HIV Nef and SIV Nef, and that this correlated with impaired PAK activation. Lastly, we identified amino acid similarity between Nef and the tumor susceptibility gene 101 and speculate that this region of Nef could be involved in late-stage events of the virus replication cycle. Taken together, these studies revealed amino acids of Nef that mediate its function, possibly by influencing the conformational state of Nef to discriminate between different subsets of ligands.