HIV-1 Nef inhibits a common activation pathway in NIH-3T3 cells.
The human immunodeficiency virus type 1 (HIV-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 biochemical 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 1,4,5-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.
De
S K
SK
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Marsh
J W
JW
D016475:3T3 Cells; D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D015693:Gene Products, nef / Q000096:biosynthesis / Q000378:metabolism*; D015754:Genes, nef; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D019868:Kanamycin Kinase; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008979:Moloney murine leukemia virus; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D054311:nef Gene Products, Human Immunodeficiency Virus; D015544:Inositol 1,4,5-Trisphosphate; D008715:Methionine; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D001839:Bombesin; D002118:Calcium