The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR

Abstract

The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8(+) T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.

Fig 1

PVR is downregulated by Nef in HIV-infected Jurkat cells. (A) Jurkat cells were not infected (n.i.) or infected with HIV-1 (NL4-3) wt or ΔNef isolates. After 3 days, expression of cell surface PVR or HLA-I was analyzed together with intracellular p24 expression by two-color flow cytometry. Values corresponding to geometric mean fluorescence intensity specific for PVR and HLA-I of p24-negative cells (left gate) and productively infected p24⁺ cells (right gate) are indicated. (B) The mean fluorescence intensity ± SE relative to PVR in n.i. and in p24⁺ wt- and ΔNef isolate-infected cells was determined as described for panel A in eight independent experiments. ***, P < 0.001. (C and D) Jurkat cells were infected for 3 days and analyzed as described for panel A. Data from one representative experiment out of three are shown. (C) An NL4-3 virus expressing the Nef protein of the SF2 strain (wt-SF2Nef) or derived from an HIV-infected patient (wt-RP4Nef) was used. (D) Cells were infected with NL4-3-based viruses in which the nef coding sequence was either conserved (wt), disrupted (ΔNef), or replaced with HIV-2 nef (wt-HIV2Nef), followed by an internal ribosomal entry site (IRES) and the gene encoding the enhanced GFP (NL4-3 nef-IRES-eGFP viruses). The mean fluorescence intensity of PVR on GFP-negative and GFP⁺ cells is reported.

Fig 2

Surface PVR expression in primary CD4⁺ T cells undergoing HIV-1 infection. (A) Purified CD4⁺ T lymphocytes isolated from a healthy donor were not infected (n.i.) or infected with HIV-1 wt or ΔNef and then stimulated with SEB 5 days later. As control, an aliquot of n.i. cells was left unstimulated (n.a.). After 3 days, cell surface PVR and intracellular p24 were analyzed as described in the legend to Fig. 1. Isotype control staining (IgG) of n.a. cells is also shown. (B) Quantitative assessment of PVR upmodulation upon activation of CD4⁺ T cells infected with wt or ΔNef virus or not infected. The modulation of PVR expression under the indicated cell conditions was calculated by subtracting from each mean fluorescence intensity the value obtained for the isotype control staining and dividing the mean fluorescence intensity obtained for n.i. and wt- and ΔNef virus-infected cells by the mean fluorescence intensity obtained for n.a. cells. Values are averages ± SEs derived from six independent experiments, as shown in panel A. *, P < 0.05; ***, P < 0.001.

Fig 3

Mechanisms of PVR modulation by HIV-1 Nef. (A) At 3 days postinfection, noninfected (n.i.) and wt- and ΔNef virus-infected Jurkat cells were permeabilized and then analyzed by two-color flow cytometry for total PVR or HLA-I levels as well as for p24 expression. Mean fluorescence intensity values are indicated. (B) Jurkat cells were not infected or infected with HIV-1 wt, ΔNef, or expressing a mutated Nef protein, as indicated, and then analyzed 3 days later for the expression of intracellular p24 and surface PVR or HLA-I, as described in the legend to Fig. 1A. Surface CD4 was analyzed at 1 day p.i. to limit the contribution of the late viral proteins Vpu and Env to the total CD4 downregulation. Surface expression of each molecule in cells infected with the indicated viruses was calculated by setting the mean fluorescence intensity obtained for n.i. cells to 100%. Values are averages ± SEs derived from at least three independent experiments. The dotted lines indicate the maximal surface expression found on wt HIV-infected cells.

Fig 4

Vpu contributes to the activity of Nef on surface PVR downregulation. (A) 293T cells were transfected with 0.5 μg of vector expressing DsRed, Nef-DsRed, or G₂A-DsRed and analyzed 48 h later by two-color flow cytometry to measure DsRed fluorescence and cell surface PVR. Mean fluorescence intensity values relative to the value for PVR in DsRed-negative (left gate) and DsRed-positive (right gate) cells are indicated. (B) Total lysates of cells described in panel A were analyzed by Western blotting with anti-Nef and antiactin antibodies. (C) Jurkat cells were infected with HIV-1 either wt or defective for the expression of Nef, Vpu, or both proteins, as indicated, and analyzed for the surface expression of PVR or CD4 (on day 3 or 1 p.i., respectively), together with intracellular p24 accumulation. (D) The mean fluorescence intensity ± SE for PVR in n.i. and in p24⁺ infected cells was determined as described for panel C in seven independent experiments. *, P < 0.05; ***, P < 0.001. (E and F) 293T cells were transfected and analyzed as described for panels A and B, with the difference that the indicated YFP, Vpu-YFP, UM2/6-YFP, or URD-YFP proteins were expressed and monitored by YFP-specific fluorescence and anti-GFP/YFP Western blotting. The YFP-tagged Vpu proteins and YFP alone are indicated with a filled arrowhead and an open arrowhead, respectively. (G) 293T cells were doubly transfected with 0.5 μg of each vector in order to express DsRed or DsRed-Nef together with YFP or Vpu-YFP and then analyzed 48 h later by FACS. The expression of PVR on gated double-positive cells is indicated. The open histogram shows unlabeled cells. Results obtained with 293T cells (A, B, and E to G) are from one representative experiment out of three.

Fig 5

Involvement of DNAM-1 in the lysis of HIV-1-infected cells. IL-2-activated NK cells were tested for cytotoxicity against noninfected (n.i.) and wt- and ΔNef virus-infected Jurkat cell targets in a FACS-based assay. (A) Saturating amounts of blocking MAb to the indicated receptors or isotype control (IgG1) were preincubated with NK cells before testing at the indicated effector-to-target cell (E:T) ratio. Data are expressed as percent lysis. Representative results from one out of three independent experiments are shown. (B) Data are expressed as percent inhibition of lysis calculated according to the formula: [1 − (% of lysis with MAb)/(% of lysis with IgG1)] × 100. Results from three independent experiments (mean ± SE) at an E:T ratio of 4:1 or 3:1 are shown.