HIV-1 NEF INHIBITS A COMMON ACTIVATION PATHWAY IN NIH-3T3 CELLS

The human immunodeficiency virus type 1 (HlV-1) Nef is a myristylated 27-kDa, cytoplasmic protein. It is attributed to have suppressive effects on LTR-based expression and T cell activation. Additionally, SIV nef has been shown to possess an essential in vivo function in the development of immunodeficiency. To define the bio chemical activity of HIV-1 Nef in a signal transduction pathway, we have transduced murine NIH-3T3 cells with a retroviral nef expression system, In nef-expressing cells, but not in controls, the proliferative response to bombesin and platelet-derived growth factor (PDGF) was eliminated. Analysis of an early signal pathway metabolite, inositol l,4,5-trisphosphate following bombesin and PDGF treatment to quiscent cells, revealed that both control and nef-transformed cells displayed similar kinetics of signal formation. Normally, inositol 1,4,5-trisphosphate mediates increase in the cytosolic free Ca2+ ([Ca2+](i)). Upon stimulation with bombesin or PDGF, control cells displayed a 2-4-fold increase of [Ca2+](i) over the basal level, while the [Ca2+](i) response in nef-expressing NIH-3T3 cells was lacking or highly diminished. However, the release of [Ca2+](i) from the intracellular store of the nef-expressing cells by an endomembrane Ca2+ ATPase inhibitor, thapsigargin, revealed that these cells contained normal Ca2+ stores. These results suggest a specific, definable biochemical activity for the HIV-1 Nef protein in the context of a well characterized cellular activation pathway. Our results thus define, for the first time, a unique function of Nef that is not limited to an alteration of T cell function or of expression of a T cell surface antigen.