HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection

Abstract

All primate lentiviruses (HIV-1, HIV-2, SIV) encode Nef proteins, which are important for viral replication and pathogenicity in vivo. It is not known how Nef regulates these processes. It has been suggested that Nef protects infected cells from apoptosis and recognition by cytotoxic T lymphocytes. Other studies suggest that Nef influences the activation state of the infected cell, thereby enhancing the ability of that cell to support viral replication. Here we show that macrophages that express Nef or are stimulated through the CD40 receptor release a paracrine factor that renders T lymphocytes permissive to HIV-1 infection. This activity requires the upregulation of B-cell receptors involved in the alternative pathway of T-lymphocyte stimulation. T lymphocytes stimulated through this pathway become susceptible to viral infection without progressing through the cell cycle. We identify two proteins, soluble CD23 and soluble ICAM, that are induced from macrophages by Nef and CD40L, and which mediate their effects on lymphocyte permissivity. Our results reveal a mechanism by which Nef expands the cellular reservoir of HIV-1 by permitting the infection of resting T lymphocytes.

Figure 1

Macrophages expressing Nef render lymphocytes permissive to HIV-1 replication. a, Assay for permissivity. The tropism HIV-1_SFI and HIV-1_LAI is restricted to macrophages and T cells, respectively. HIV-1_LAI production (detected by titration on MT4 cells) provides an indication that those lymphocytes were rendered permissive for viral replication by infected macrophages. b, Wild-type but not Nef-deleted viruses induce lymphocyte permissivity. Virus production from HIV-1 _SFI WT- and ΔNef-infected macrophages in the absence of lymphocyte culture (left panel); MT4 titration of HIV-1_LAI in supernatants of lymphocytes co-cultured with HIV-1_SFI WT-infected, HIV-1_SFI ΔNef-infected and mock-infected macrophages (right panel). c, CD40 stimulation induces lymphocyte permissivity. Viral replication was examined in resting lymphocytes exposed to supernatants from Nef- and GFP-expressing macrophages, or from CD40L-stimulated macrophages. d, Inhibition of Nef- and CD40-induced permissivity by an IκBα ‘super-repressor’. Macrophages were subject to CD40L stimulation/adenovirus infections as indicated, and supernatants were examined for induction of permissivity. RT, reverse transcriptase.

Figure 2

Nef and CD40 promote resting T-lymphocyte infection by regulating co-stimulatory receptors on B lymphocytes. a, Nef- and CD40-mediated permissivity requires the presence of B lymphocytes. B lymphocytes were incubated with supernatants from Nef-expressing or CD40L-stimulated macrophages. After 72 h, the B cells (Bc) or B-cell supernatants (Bs) were added to autologous lymphocytes, which were then examined for susceptibility to HIV-1 infection. b, Nef and CD40 upregulate similar receptors on B cells. B lymphocytes were incubated with supernatants from Nef- or GFP-expressing or CD40L-treated macrophages, and expression of CD22, CD54 and CD58 on CD19⁺ B cells was determined by FACS. c, B-lymphocyte signals induce cell-cycle-independent infection of T lymphocytes. Following ligation of the indicated receptors, resting CD3⁺ T lymphocytes were infected with HIV-1_HSA and the level of infection and cell-cycle status determined by FACS (upper panel) and by thymidine incorporation (lower panel), respectively. Rabbit F(ab)₂-cross-linked mouse IgG (isotype) is shown as a control. d, Expression profiles of cell-cycle regulatory genes in antibody-stimulated lymphocytes. Purified CD3⁺ lymphocytes were stimulated as in c and transcript levels for the indicated genes determined by RNase protection assay. e, Transcript profiles of cellular activation markers. f, Viral replication profiles in antibody-stimulated T lymphocytes. CycE, Cyclin-E; Cyc-D, Cyclin-D; MHC, major histocompatibility complex.

Figure 3

Identification of Nef and CD40-regulated B-lymphocyte receptors that overcome blocks to virus production. a, Nef and CD40 upregulate CD80/CD86 expression on B lymphocytes. B lymphocytes were immunophenotyped following incubation with supernatants from Nef- or GFP-expressing on CD40L-stimulated macrophages. b, Antibody to CD80 blocks induction of permissivity by Nef and CD40. Supernatants of Nef-expressing and CD40L-stimulated macrophages were incubated with autologous, resting lymphocytes in the presence of αCD80, αCD86 or isotype (Iso) antibodies, then examined for the ability to support HIV-1 replication. c, The additional CD80 stimulus induces cell-cycle progression. The isotype control comprises Rabbit F(ab)₂-cross-linked murine IgG., The additional CD80 stimulus reconstitutes a productive infection.

Figure 4

Induction of lymphocyte permissivity by Nef and CD40 is mediated by sCD23 and sICAM. a, Nef and CD40 induce sCD23 and sICAM release from macrophages. Replication profiles for wild-type and ΔNef viruses are indicated along with levels of sICAM, sCD23 and MCSF in wild-type-infected, ΔNef-infected and uninfected macrophage cultures. Results are representative of three independent donors. Right panels, sICAM and sCD23 levels in supernatants of macrophages 18 h after transduction with a Nef adenovirus vector or after CD40L stimulation. Levels in adeno-GFP-infected and mock-infected cultures are shown for comparison. b, Effects of sCD23 and sICAM immunodepletion on induction of permissivity by Nef or CD40L. Left panel, levels of viral replication. Right panels, levels of sICAM and sCD23 in immunodepleted macrophage supernatants. Supernatants from adeno-GFP-transduced and mock-infected cultures serve as controls. c, sICAM and sCD23 upregulate receptors on B lymphocytes. Purified B lymphocytes were immunophenotyped after incubation with recombinant sICAM (50 ng ml⁻¹) or sCD23 (75 ng ml⁻¹). Cells were gated on CD19⁺ and CD58⁺ B cells for CD22/CD58 and CD80/CD86 analysis, respectively. d, sCD23 and sICAM promote T-lymphocyte permissivity. T lymphocytes were incubated with sICAM, sCD23 or isotype-treated B cells then infected with HIV-1_HSA. Infected resting (${\text{HIV}}_{\text{HSA}}^{+}∕\text{Ki}{67}^{-}$) and cycling ($\text{HIV-}{1}_{\text{HSA}}^{+}∕\text{Ki}{67}^{+}$) T lymphocytes were identified by FACS (left panels). Levels of virus replication in T-lymphocyte cultures stimulated with sCD23, sICAM or isotype-treated B cells (right panel).

Figure 5

Mechanistic model for induction of lymphocyte permissivity by Nef and CD40. Physiological stimulation of CD40 in macrophages leads to NF-κB activation. This signalling pathway is intersected by HIV-1 Nef. In macrophages, activation of CD40 or expression of Nef induces the release of sCD23 and sICAM, which upregulate the expression of co-stimulatory receptors on B lymphocytes. These, in turn, interact with their corresponding ligands on T lymphocytes, rendering them permissive to HIV-1 infection. The types of receptor that are upregulated on B cells dictate the outcome of the infection. The induction of CD22 and CD58 on B cells is mediated primarily by sCD23. The action of these receptors on T cells does not lead to T-cell proliferation, but is sufficient to permit virus entry and de novo expression of viral proteins, but not virion release. Induction of CD80 on B cells, as mediated primarily by sICAM, provides signals that promote entry of T cells into the cell cycle, thereby allowing the productive infection of these cells.