Abstract
nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.
Highlights
Human HIV11 is a complex retrovirus containing several genes which regulate viral replication and gene expression. nef is one of the seven nonstructural genes highly conserved in HIV2, and SIV
(i) purified Nef protein microinjected in peripheral blood lymphocytes inhibits the proliferative response to IL-2 [9]; (ii) constitutive expression of nef inhibits signaling by IL-2 and TCR in leukemic cells [9, 12,13,14,15] and by growth factors in murine fibroblasts [16]; (iii) transgenic expression in thymocytes interferes with TCR signaling and perturbs their development [8, 17]
We investigated the interactions between the Nef protein and the signaling pathways triggered by the platelet-derived growth factor (PDGF) receptor, which are among the best characterized [18]
Summary
Antibodies, and Cells—HIV1-nefBRU full-length cDNA was kindly provided by Dr Montaigner, anti-Nef and anti-PDGF receptor antibodies by Dr Samuel and by Dr Heldin. PI 3-kinase activity was assayed on the immunoprecipitates in the presence of [␥-32P]ATP and phosphoinositides (Sigma), as described previously [19]. In these conditions, PI 3-kinase phosphorylates preferentially PI[4,5]P2, generating PI[3,4,5]P3 [20]. Proliferation and Transwell Migration Assay—Cells were seeded in 24-well plates (105 cells/well) and incubated in DMEM/1% FCS alone or containing either increasing concentrations of PDGF or 10% fetal calf serum. One ml of DMEM/1% FCS, alone or containing the stimulating factor, was added to the lower compartment. Cells migrated to the lower side of the filter were fixed, stained as above, and solubilized in 10% acetic acid.
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