HIV Type 1 Nef Increases the Association of T Cell Receptor (TCR)-Signaling Molecules with T Cell Rafts and Promotes Activation-Induced Raft Fusion

Abstract

HIV-1 Nef (Nef) is a myristoylated protein that contributes to HIV disease pathogenesis. Nef has a modulatory effect on T cell receptor (TCR) signaling, resulting in up-regulation of interleukin-2 (IL-2) production in stimulated T cells. Recent studies have shown that efficient TCR signaling requires enhanced association of TCR-signaling molecules with plasma membrane microdomains (lipid rafts) and fusion of rafts into larger structures. We utilized Jurkat T cell lines expressing wild-type Nef (Nef_wt) and a myristoylation-deficient form of Nef (Nef_G2_A), from an inducible promoter, to determine the effects of Nef on the association of TCR-signaling molecules with rafts in nonstimulated T cells. In addition, the effect of Nef on raft size, before and after TCR activation by CD3 cross-linking, was also examined. Following induction, Nef_wt was associated with both rafts and nonrafts, while Nef_G2_A was almost exclusively cytosolic. Induction of Nef_wt, but not Nef_G2_A, coincided with an increased association of the src family tyrosine kinase, Lck, and TCRζ with rafts, but not with nonrafts. Further, rafts were found to be significantly larger in CD3-activated T cells in the presence of Nef_wt when compared to nonexpressing cells. We propose that myristoylated, raft-localized Nef primes resting T cells for activation by increasing the levels of signaling molecules within rafts, and that TCR activation is enhanced by the capacity of Nef to promote raft fusion.