The Nef protein of HIV-1 associates with rafts and primes T cells for activation

Abstract

The Nef protein is an important virulence factor of primate lentiviruses, yet the mechanisms by which it exerts this influence are imperfectly understood. Here, using an inducible system, we demonstrate that Nef increases IL-2 secretion from T cells stimulated via CD3 or CD28. This effect requires the conservation of the Nef myristoylation signal and SH3-binding proline-based motif. Together with several proteins involved in the initiation and propagation of T cell signaling, Nef associates with membrane microdomains known as rafts. The Nef-mediated superinduction of IL-2 reflects the activation of both NFAT and NFkappaB. Accordingly, Nef also enhances HIV-1 transcription in response to CD3 or CD28 stimulation. Nef-induced IL-2 hyperresponsiveness is also observed in primary CD4 lymphocytes. Overall, these data suggest that Nef acts at the level of rafts to prime T cells for activation. Likely consequences of this effect are the promotion of HIV-1 replication and the facilitation of virus spread.

Figures

Figure 1

Characterization of Nef-inducible Jurkat T lymphoid cell lines. (A) Nef-specific Western blot analysis of indicated cells grown in the presence or absence of tetracycline for 5 days. Nef levels in nef-induced (+), control (−), and HIV-1-infected (HIV) Jurkat cells are compared underneath. (B) Cell surface levels of CD4 and MHC-I measured by fluorescence-activated cell sorter analysis. Result is representative of the two clones tested in each case.

Figure 2

Nef enhances IL-2 production in response to CD3 or CD28 stimulation. (A and B) Uninduced (open bars) or induced (solid bars) cells were stimulated with the indicated antibodies, with crosslinking for CD3. IL-2 concentration in the supernatant was measured by ELISA 16–18 h later. Without stimulation, no IL-2 was detected (not shown). In C, results are expressed as the ratio of IL-2 values measured in the supernatants of induced vs. uninduced cells. Data are averages from representative duplicate experiments.

Figure 3

Myristoylation-dependent association of Nef with DIG microdomains. (A) Equal volumes from the DIG- and Triton X-100 (Tx)-soluble fractions, or diluates from the latter, were subjected to SDS/PAGE. Electrophoresis was stopped just after the dye front entered the separating gel, and proteins were revealed by Coomassie blue staining. (B) Equal volumes from DIG- (lane 1) and Tx-soluble (lane 3) fractions, or a 1:30 dilution of the latter (lane 2), were analyzed by Western blotting with indicated antibodies. Two distinct species of Nef (arrows) are detected in DIG- but not Tx-soluble fractions. (C) Nef-specific Western blot analysis of DIG- and Tx-soluble fractions from control and nef-expressing cells, loading equal amounts of protein in all cases. The two species of Nef (visible in B) are not clearly separated on this low resolution gel. (D) Phosphotyrosine-specific Western blot analysis of DIG fractions of control (−) and nef-expressing (+) cells.

Figure 4

Nef potentiates IL-2 promoter activation. Cells grown 4 days in tetracycline-free medium were electroporated with an IL-2 promoter luciferase reporter plasmid and were kept either with (open bars) or without (dark bars) tetracycline. Forty-eight hours later, aliquots of cells from each group either were left untreated or were stimulated as indicated (with crosslinking for α-CD3) for fourteen hours before measuring luciferase activity. Fold activation represents the ratio of values in test sample vs. its uninduced/unstimulated control. Data are averages from representative duplicate experiment, with variability as error bar. Without stimulation, Nef had no effect (not shown).

Figure 5

Nef superinduces NFAT and NFκB activities in response to CD3 or CD28 stimulation. NFAT- (Top), NFκB- (Middle), and AP-1- (Bottom) luciferase plasmids were transfected into indicated cells, using a procedure similar to that described for Fig. 4 (without crosslinking). Without stimulation, Nef had no effect (not shown). Values are averages of two independent transfections. Open bars, uninduced cells; solid bar, induced cells.

Figure 6

Nef enhances HIV-1 promoter responsiveness to CD3 or CD28 stimulation. Wild-type or NF-κB-mutated HIV-1 LTR luciferase plasmids were transfected into control or Nef-producing Jurkat cells. Other experimental conditions were the same as for Fig. 4 (without crosslinking). Values are averages of two independent transfections. Open bars, uninduced cells; solid bar, induced cells.

Figure 7

Nef increases NFAT and HIV-1 LTR transcriptional responsiveness in transiently transfected Jurkat T cells. D4 Jurkat T cells were cotransfected with NFAT (A) or HIV-1 LTR (B) luciferase plasmids and either an empty vector (pTet-Splice) or the indicated Nef expression plasmids, plus pRL-TK as an internal control. Twenty-four hours later, cells either were left unstimulated or were treated with 3 μg/ml anti-CD3 or CD28 antibodies for fourteen hours and were analyzed for luciferase activity. Ratios of values obtained with vs. without stimulation are shown, averaging two independent transfections.