The HIV regulatory protein nef has been demonstrated to have multiple effects on HIV replication in vitro. These include both an inhibitory action on HIV-replication through the down-regulation of CD4 expression, and an enhancement of viral replication in certain systems through multiple mechanisms. In one system, nef in trans increased CD4+ T-cell activation and viral replication. In the macaque model, a nef-deleted simian immunodeficiency virus (SIV) has been found to be non-pathogenic; when used as a vaccine it was protective against infection with pathogenic SIV. Similarly, certain HIV-infected individuals identified with a mild or non-progressing course of HIV disease have been found to harbor nef deletions in their virus. Thus, nef may function as a virulence factor, possibly through activation of target cells. The purpose of this project is to evaluate the effect of HIV nef on the ability of HIV to use the chemokine co-receptors for entry, signaling, and activation of target cells. A model is proposed whereby HIV nef both decreases surface expression of CCR5 and increases the ability of M-tropic gp160 to signal through CCR5 and activate target cells. The effect of nef on surface expression and signaling capacity of the HIV co-receptors, CXCR4, CCR3, CCR2b and CCR5, will be analyzed. Peripheral blood mononuclear cells (PBMCs) will be the principal cell population studied. We have observed that nef, delivered either by transfection or vaccinia virus to PBMCs, can down regulate the expression of CD4 and CCR5. MIP-1beta signaling through CCR5 was observed not to be decreased in the setting of less surface expression of CCR5. This suggests a differential interaction between nef, CD4, and HIV co-receptors that may lead to a possible enhancing activity on CCR5 signaling by interacting with the signaling pathway, and a reduction of surface expression to decrease superinfection.