Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.
Blotting, Western
,CD4-Positive T-Lymphocytes
,Cell Cycle Proteins
,Cell Line
,Enzyme-Linked Immunosorbent Assay
,Gene Products, nef
,Guanine Nucleotide Exchange Factors
,HIV
,Humans
,Immunoprecipitation
,Membrane Microdomains
,Microscopy, Confocal
,Proto-Oncogene Proteins c-cbl
,RNA, Small Interfering
,Rho Guanine Nucleotide Exchange Factors
,Signal Transduction
,Ubiquitin-Conjugating Enzymes
,Virus Replication
,cdc42 GTP-Binding Protein
,nef Gene Products, Human Immunodeficiency Virus