Nef is an accessory protein encoded by human and simian immunodeficiency viruses (HIV and SIV), and is critical for viral pathogenicity in vivo.The structure of Nef has been resolved and the major cellular activities of Nef are generally described as down-regulation of cell surface molecules, enhancement of virus infectivity and regulation of cell signaling and activation. Macrophages represent a key target of HIV-1 infection and may contribute significantly to viral pathogenesis by facilitating viral propagation, maintaining a viral reservoir and regulating viral replication. During HIV-1 infection, various cytokines and chemokines are induced for viral advantages more than for host defense. We have previously demonstrated that HIV-1 Nef regulates the release of chemokines, MIP-1α and MIP-1ß, from infected macrophages and have proposed that this may enhance conditions for viral replication by promoting recruitment of substrate lymphocytes to sites of infection (1). However, the molecular basis for this Nef activity remains to be defined. The main goals of this thesis are to identify the functional motif in Nef that is responsible for chemokine induction in macrophages and to elucidate the relevance of this motif to other Nef functions. Using a mutagenesis approach, we have eventually identified a novel motif (KEK) that regulates chemokine production in infected macrophages after we excluded several previously described Nef motifs. This motif is conserved in both HIV-1 and SIV Nef proteins. Mutations in this domain abrogated MIP-1ß induction as well as the Nef-dependent release of other secretory factors by macrophages. However, disruption of this motif did not affect other Nef-ascribed activities such as CD4 and MHC-I down-regulation. In addition, we have determined the involvement of viral Env proteins in Nef-induced chemokine production. Distinct signaling pathways that regulate chemokine release in macrophage will also be described. Finally, several possible roles of the KEK motif are proposed and some preliminary results of co-immunoprecipitation experiments will be presented which aim to characterize cellular proteins involved in chemokine regulation by Nef. Collectively, our studies reveal a specific determinant within Nef that is critical for chemokine release by Nef. Identification of this motif paves the way for future studies to explore the molecular machanisms of Nef-regulated cell signaling pathways. Such knowledge may point to new therapeutic strategies that interrupt Nef function and limit the course of HIV-1 infection.