HIV-1 infection leads to a state of chronic immune activation and progressive deterioration in immune function. The loss of CD4+ T lymphocytes plays a key role in disease progression. Although several hypotheses concerning the CD4+ T lymphocytes depletion have been postulated, the underlying mechanisms of this specific cell depletion are still debated. We showed that NKp44L, a cellular ligand of the natural cytotoxicity receptor NKp44, is specifically induced on CD4+ T cells during HIV-1 infection and that its expression is strongly correlated with the depletion of CD4+ T cells and increased viral load . CD4+ T cells expressing NKp44L are highly sensitive to lysis mediated by activated NKp44+ NK cells [1,2]. NKp44L is specifically induced by the highly conserved 3S-motif of the HIV-1 gp41 envelope protein . More recently, we showed in macaque model, that anti3S immunization strongly decreases NKp44L expression on CD4+ T cells, and inhibits the subsequent decline in CD4 cell count after SHIV-infection . Together, these results strongly suggest that HIV-1 has acquired the ability to use NK cells to disarm the host immune system by selectively triggering CD4+ T cells killing.
The importance of NK cells in viral infection is perhaps best supported by the fact that several persistent viruses, such as cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella zoster virus developed strategies to counter recognition by NK cells [5,6]. The role of NK cells in the protection and control of HIV infection is currently unclear. A recent whole genome association study identified SNPs, which have an impact on NK function, and appear to be important for host control of HIV infection, one which is associated with HLA-B57 and the second correlates with higher HLA-C mRNA expression . In addition, Martin et al.  have shown an innate partnership of HLA-B and KIR3DS1, delaying the progression to AIDS. Concomitantly, viral persistence is achieved by the combined activity of viral latency gene expression and the production of immune-evasion molecules that help the virus to elude detection by NK cells. Studies of these viral immune modulators have contributed to an improved understanding of the intricate relationship between pathogens and their hosts. Such evasion strategies include expression of viral homolog of MHC class-I, which block NK cell-mediated killing, selective modulation of MHC class-I expression to increase inhibition of NK cells; interference with cytokine or chemokine pathways involved in NK cell activation and antagonism of NK cell activation receptors and their ligands on target cells . Like other viruses, HIV-1 has developed various strategies to block NK cell activity. First, HIV-1 induces a selective modulation of MHC class-I allele expression mediated by Nef protein, which accelerates the endocytosis of class-I molecules from the cell surface . Cohen et al.  showed that the selective downmodulation of HLA-A, and HLA-B, but not HLA-C and HLA-E on the cell-surface render infected cells resistant to NK-cell killing. In line with these data, Bonaparte et al.  have shown that purified peripheral blood NK cells cannot kill autologous T cells blasts efficiently, despite downmodulation of MHC class-I molecules. Concomitantly, it has also been suggested that HIV-1 Tat protein can block NK cell activation and function [13,14].
In this study, we sought to determine the mechanism that HIV has evolved to avoid detection by NK cells through NKp44L expression. We discovered that during HIV-1 infection, uninfected CD4+ T cells exclusively expressed NKp44L. We showed that the HIV-1 pathogenicity factor Nef mediates NKp44L downmodulation in HIV-infected cells. Nef uses a pathway different from those previously described for the downregulation of CD4 molecule and MHC Class-I molecules. This study represents a novel illustration of the crucial role played by Nef in the evasion of infected cells from NK cells.
HIV-infected progressor and long-term nonprogressor (asymptomatic long-term) individuals
Long-term slow progressor patients were obtained from the French asymptomatic long-term (ALT) group cohort. These patients were defined at enrollment by a stable (>8 years) CD4 cells count (>600/μl) in absence of antiretroviral therapy . Peripheral blood mononuclear cells (PBMC) from untreated HIV-infected progressor patients kindly provided from Prof Jack Leibowitch (Hôpital Raymond Poincaré, Garches, France) were obtained after approval by the relevant institutional review board. All individuals provided written informed consent. Routine laboratory analysis of these patents included viral load determination, and complete blood count.
CD4+ T cells purification and culture
Leukocytes from healthy donors were obtained by buffy coat from the hospital blood bank (Hôpital Pitié-Salpêtrière, Paris, France). CD4+ T cells were purified using CD4 microbeads (Miltenyi). Flow cytometric analysis demonstrated a purity of more than 95% CD4+ cells. Purified CD4+ T cells were activated with 1 μg/ml phytohemagglutinin (PHA)-L (Murex, Chatillon, France) in RPMI-1640 medium supplemented with 10% FCS, and then cultured with 10 IU/ml IL2 (Roche), for an additional 2 days period.
Peptides and antibodies
The synthetic NH2-PWNASSWSNKSLEQIW-COOH 15-mer peptide, called 3S, from gp41 HIV-1 protein was purchased from Covalabs (Villeurbanne, France). Purity was more than 80%, as verified by HPLC profile. Monoclonal anti-NKp44L antibody (IgM; #7.1) was previously described .
Viral stock generation and HIV infection
Virus stocks were generated by transfection of proviral plasmids into 293T cells by the calcium-phosphate method. Two days after transfection, culture supernatants were harvested and concentration of virus stocks determined, as previously described .
HIV-1 infection of purified CD4+ T cells was carried out with 50 ng of virus per 106 cells for 2 h at 37°C in presence of 20 μg/ml DEAE dextran. After incubation, cells were extensively washed and then cultured in presence of 10 IU/ml IL2 for 7 days.
HIV-1 Nef mutants
Isogenic HIV-1 proviral clones encoding for established Nef mutants have been previously described . Briefly, the prototype chimera containing the HIVNL4-3 provirus with the HIVSF2 nef gene was designated wild type, the HIVNL4-3 provirus deleted to the nef gene was designated Δnef, and the respective nef mutants were all designated according to their amino acid changes in the encoded Nef protein; including G2A (mutation G2A), AxxA (mutation P76/79A), and LLAA (mutation L168/169A), as previously described .
Recombinant vaccinia virus expressing HIV-1 proteins
CD4+ T cells were infected at a multiplicity of infection of 20 plaque-forming units (pfu)/cell with wild-type vaccinia virus or with recombinant vaccinia viruses expressing various HIV-1 proteins including Gag, Pol, gp120-Env, gp41-Env, Tat, Vif Vpu, Vpr, Nef, and Δ145–200 Nef, as previously described . Recombinant vaccinia viruses for HIVBRU proteins were provided from Transgene (Strasbourg, France). The efficacy of vaccinia infection was verified with antivaccinia polyclonal Ab (AB Technologies).
PCR amplification and sequencing of nef gene
DNA was extracted by means of the DNA extraction kit protocol QIAEXII from PBMCs obtained from the ALT individuals. This DNA was directly submitted to a nested PCR amplification procedure targeted to nef gene. The amplified DNA products were purified by electrophoresis on a 1.8% TBE agarose gel, and nucleotide sequence was determined on both strands as previously described . Nucleotide sequences were aligned with the CLUTAL W software and integrated in GenBank database under accession numbers: AY856760, AY856765, AY856766, AY856768, AY856772, and AY856777 for 04.008, 04.048, 04.061, 05.002, 09.016, and 11.003 LTNP samples, respectively.
Flow cytometric analysis
FACS analysis was performed on purified CD4+ T cells or PBMC. Isotype-matched immunoglobulin served as the negative control. Cells were incubated with 2 μg anti-NKp44L mAb (#7.1)  for 1 h at 4°C, washed in PBS/1% BSA and then incubated with 1: 100 PE-conjugated antimouse IgM mAb (BD Biosciences, Le Pont de Claix, France) plus APC-conjugated antihuman CD4 mAb (Beckman Coulter, Villepinte, France), for 30 min at 4°C. These cells were fixed and permeabilized using cytofix/cytoperm kit (BD Biosciences), according to the manufacturer's instructions, and then stained using anti-HIV-1 p24 mAb (KC57, Beckman Coulter, Villepinte, France) for 1 h at 4°C. At least 20 000 leukocytes were detected on a FACScalibur (BD Biosciences, Le Pont de Claix, France). Results were analyzed with cellQuest software (BD Biosciences, Le pont de Claix, France) and expressed as percentage of CD4+ T cells in the mononuclear cells gate.
Cytolytic and degranulation assays
Purified NK cells were culture in presence of 1000 IU/ml IL2 (Proleukin; Chiron France, Suresnes, France), for 5 days. Their cytolytic activity was assayed in a standard 4-h 51Cr-release assay. The role of NKp44L was analyzed by adding an anti-NKp44L (#7.1) mAb or its IgM isotype control (BD Biosciences, Le pont de Claix, France), at a final concentration of 20 μg/ml, and preincubating them with the effector cells for 30 min at 37°C, as described . Some functional experiments were realized in presence of specific inhibitors of the myristoylation; DL-β-hydroxymyristic acid (2 OHM) (Sigma Aldrich, Saint-Quentin Fallavier, France) or 4-oxatetradecanoic acid (4 OXA) (Sigma Aldrich) at 100 μmol/l for 48 h. Cell viability, and intracellular expression of p24 antigen were tested before the 51Cr release assays, revealing no side effect on CD4+ T cells.
The degranulation assay was performed by CD107a detection, according to methods previously described . Effector cells were incubated overnight with IL2 and then resuspended at 106 cell/ml with target cells at a 1: 1 ratio in the presence of anti-CD107a mAb (BD Biosciences, Le Pont de Claix, France). After 1 h of incubation, monensin (Sigma Aldrich, Saint-Quentin Fallavier, France) was added at 6 μg/ml for an additional 4 h incubation. Expression of CD107a was detected by flow cytometry following NK cell straining with anti-CD3, anti-CD56, and anti-NKp44 mAb (Beckman Coulter, Villepinte, France). Results were expressed as percentage of CD3-CD56+ NK cells.
Expression of NKp44L on HIV-infected CD4+ T cells
Previously, we have shown that NKp44L, the cellular ligand of NKp44, was overexpressed on CD4+ T cells from HIV-infected patients . In the present study, we asked whether both productively infected and noninfected CD4+ T cells expressed NKp44L. For this purpose, purified CD4+ T cells were infected with two different strains of HIV-1 (AD8 and NL4-3). Productively infected cells were visualized by intracellular Gag p24 staining. As shown in Fig. 1a, NKp44L was not detected in absence of infection. As expected, it was observed in presence of 3S-peptide from HIV-1 gp41 . When CD4+ T cells were infected with either HIVNL4-3 or HIVAD8, 30.0–48.5% of them expressed NKp44L. Interestingly, NKp44L was only expressed on bystander noninfected CD4+ T cells. Similar results were observed by Ward et al.  using NKp44-Ig fusion protein to detect NKp44 ligand on purified CD4+ T cells infected with either HIVSF162 or HIVSF128A. To avoid the possible bias due to in-vitro experiments with a limited number of HIV-1 isolates, expression of NKp44L was next investigated on CD4+ T cells from HIV-infected patients expressing different viral loads, following PHA-activation. In line with the in-vitro experiments, NKp44L was highly expressed on uninfected CD4+ T cells from HIV-infected patients, ranged from 38.2 to 57.7%, contrasting with its rare expression on p24+-infected CD4+ T cells (less than 2.4%), independently of the patient tested (Fig. 1b). Together, these data demonstrate that unlike uninfected cells, HIV-infected CD4+ T cells do not express NKp44L at their cell surface suggesting a viral escape mechanism protecting HIV-infected cells from NK cells attacks.
Nef prevents NKp44L surface expression
To obtain further insight in the role of HIV-1 proteins in the modulation of cell-surface expression of NKp44L, a panel of recombinant vaccinia virus expressing viral proteins was tested on CD4+ T cells treated with or without 3S-peptide. As Fig. 2 shows, in presence of 3S-peptide, the cell surface expression of NKp44L was practically abolished in CD4+ T cells expressing the Nef protein, but not in presence of vaccinia virus expressing Nef-deleted protein. As expected, gp41 induced an overexpression of NKp44L even in absence of 3S-peptide, as previously described . None of the other HIV proteins tested appeared to influence the expression of NKp44L.
To confirm the involvement of Nef on NKp44L downmodulation, CD4+ T cells were infected with wild type and Δnef viruses and stained for extracellular NKp44L expression and intracellular HIV p24 antigen. Similar level of cell-surface expression of NKp44L was obtained in samples infected either with wild type or Δnef HIV infectious particles (data not shown). However, NKp44L quantification on p24+-infected cells, revealed a highly significant increased, upto 12-fold, after infection with Δnef-virus particles, compared with infection with wild type virus (Fig. 3a and b).
Myristoylation of Nef is required to modulate NKp44L expression
In order to gain insight into the molecular basis of the earlier-mentioned escape mechanism from NKp44+ NK-cell recognition mediated by Nef, a panel of isogenic HIVNL4-3 proviral clones encoding established Nef variants with mutations at various amino acid residues was tested. Nef uses distinct domains to downmodulate HLA class-I and CD4 receptors. However, a N-terminal myristoylation signal, required for Nef localization at the plasma membrane, has been shown to be critical for all Nef activities [18,19]. Viruses including Nef gene with mutations that abolish N-terminal myristoylation (G2A), the capacity to downmodulate HLA class-I (P76/79A) or CD4 molecules (L168/169A) were tested. When compared with wild type, both P76/79A and L168/169A Nef mutants induce similar profiles of NKp44L on infected CD4+ T cells; indeed, the frequency of p24+-infected cells expressing NKp44L remained closed to the baseline. By contrast, the nonmyristoylated G2A mutant increased the number of p24+-infected CD4+ T cells expressing NKp44L, up to 9.9-fold, as observed with Δnef-infected cells (Fig. 3a and b).
To properly determine the mechanism of Nef mediated downregulation of NKp44L, sequencing of nef gene has been realized in long-term nonprogressor (LTNP) individuals from the French ALT cohort . In these individuals, six independent nef sequences revealed some mutations or deletions in the domains known to interact with components of the endocytic and sorting pathways [19,20]. PBMC from these LTNP patients were activated with PHA, and then culture with IL2 for 2 days to test the capacities of their CD4+ T cells to coexpressed NKp44L and HIV Gag p24. Individuals 5.002 and 11.003 both present deletion (Δ152–156 and Δ155–160, respectively) in the Nef domain, which interacts with β-cop. The individual 04.008 possesses two point mutations C55S/A56P, in the cleavage site by the viral protease. The last individual 04.048 presents two point mutations and a deletion (E62/63G & Δ65) within the EEEE domain, crucial for MHC-I downmodulation . Although the four earlier-mentioned individuals present nef sequences with small deletions or point mutations, their profiles remain similar to the one observed in wild type-infected cells (Fig. 3c). In contrast, in patients showing an eight amino-acids insertion in position 25 (04.061) or a large deletion (Δ54–100 for 09.016) in Nef, a significant expression of NKp44L was observed in p24+-infected cells (Fig. 3c), closed to the level previously observed in Δnef-infected cells (Fig. 3a). Interestingly, in those samples deficient in Nef, noninfected cells expressing NKp44L were not detected; suggesting that activated NKp44 NK cells very efficiently and preferentially destroyed this specific cell subset. Together, these data suggested that Nef mediates NKp44L intracellular retention using a nonclassical pathway.
Nef protects HIV-infected CD4+ T cells against autologous natural killer cell cytotoxicity
We next examined the cytolytic activity of autologous NK cells against CD4+ T cells infected with either wild type or ΔNef viruses. To this end, we first tested the degranulation capacities of NK cells by measuring the cell-surface mobilization of CD107a. This marker was around four-times more frequently detected on NK cells cultured with ΔNef-infected cells, when compared with wild type-infected, while being nearly absent in NK cells tested with uninfected cells (Fig. 4a). These data were confirmed using 51Cr release assay. Autologous IL2-activated NK cells were poorly efficient in lyzing uninfected as well as wild type-infected CD4+ T cells (Fig. 4b). By contrast, after infection with Δnef-HIV particles, CD4+ T cells were far more susceptible to NK cells killing. Interestingly, pretreatment of CD4+ T cells with an anti-NKp44L mAb significantly reduced their cytolytic susceptibility to NK-mediated lysis (Fig. 4b). After having shown a differential expression of NKp44L in presence of Nef variants, it was important to assess if this could affect their sensitivity to NK lysis. We thus evaluated the ability of IL2-actived NK cells to kill autologous CD4+ T cells infected with the same panel of G2A, P76/79A, and L168/169A Nef variants. As shown in Fig. 4a, a significant increase of CD107a surface expression was observed with G2A-infected or Δnef-infected CD4+ T target cells, and not with wild type-infected cells. The P76/79A or L168/169A Nef mutants behave like the wild type protein (Fig. 4a). These observations were confirmed in the killing 51Cr release assay. Indeed, NK poorly killed autologous CD4+ T cells infected with wild type, P76/79A or L168/169A Nef mutants. This cytolysis was not significantly decreased when cells where treated with anti-NKp44L mAbs (Fig. 4b). In contrast, with CD4+ T cells infected with G2A variant, the lysis significantly rises to levels observed with Δnef-infected cells. This effect was virtually abrogated when target cells were specifically neutralized with the anti-NKp44L mAb, which then behave like wild type-infected cells (Fig. 4b). This suggests that the inhibitory effect of Nef on the NK cytotoxicity mainly depends on the NKp44L pathway.
To confirm the major effect of the myristoylation of Nef on the susceptibility of HIV-infected cells through the expression of NKp44L, we finally tested the effect of two specific inhibitors of the myristoylation, the DL-β-hydroxymyristic acid (2 OHM) and the 4-oxatetradecanoic acid (4 OXA), on the wild type-infected cells sensitivity to NK killing. As shown in Fig. 3c, lysis of HIV-infected CD4+ T cells was highly increased in presence of 2 OHM and 4 OXA. More interestingly, this enhanced effect, is practically abrogated and return closed to the baseline following treatment with neutralizing anti-NKp44L mAb (Fig. 4c). Taken together, these results indicate that Nef downmodulates NKp44L and, as a result protects p24+-infected CD4+ T cells against NK cell lysis.
The present work reveals that the Nef protein of HIV-1 is responsible for the downmodulation of NKp44L in infected CD4+ T cells. Other viruses are known to manipulate the sensitivity of infected cells to NK lysis. For instance, infection with CMV leads to the upregulation of expression of NKG2D ligands. Opposing this effect, CMV encodes proteins that interfere with ligand expression at the cell surface, like UL16, which retains MICA and ULPB, two NKG2D ligands in an internal compartment [21–23]. Similarly, Thomas et al.  have recently observed that K5, a Kaposi's sarcoma-associated herpes virus (KSHV) protein, protects infected cells against NK cell cytotoxicity by downmodulating NKG2D (MICA, MICB) and NKp80 (AICL) ligands. This strongly suggests that downregulation of activating ligands by some viruses are a powerful means of evasion from NK cell antiviral functions. As currently described, the immune response to viral infections comprises a complex interplay between host cells and virus-infected cells. The main immune effector cells in antiviral response are NK cells and cytotoxic T lymphocytes, which secrete inflammatory mediator molecules and directly lyze infected cells. NK cells express multiple cell surface receptors that do not undergo rearrangement. NK cell surface displays both activating and inhibitory receptors, which recognize different determinants on the surface of infected cells. It is the integration of signals that are perceived by these NK cell surface receptors that determines NK cell activity [25,26].
Interestingly, pathogenic viruses counter host antiviral responses by expressing specialized genes. For instance, the HIV-1 Nef molecule, a 27-kDa myristoylated protein, plays a central role in pathogenesis. It is one of the first viral proteins expressed following infection. This multifunctional protein enhances virus replication and promotes immune evasion of HIV-infected cells by manipulating transport and signaling pathways. Nef downmodulates MHC class-I and MHC class-II molecules, thereby helping the virus to evade both CD4+ and CD8+ T-cell specific responses. In rhesus macaques, the downmodulation of MHC class-I by SIV Nef limits CD8+ T-cell-mediating killing and contributes to the pathogenic effect of Nef . Macaques infected with a SIV ΔNef do not progress rapidly to disease . Nef is required to maintain high numbers of infected cells in lymph nodes, indicating that in vivo, the presence of this viral protein directly affects the localization, mobility and fate of infected cells . Furthermore, Nef triggers CD4 molecule downregulation, which has a profound impact on the fitness of viral progeny. Nef also ably modulates specific processes such as apoptosis, cell activation, and signal transduction [19,20,29]. One of the striking illustrations of the pathogenic potential of Nef was the description of long-term nonprogressor patients, harboring aberrant HIV strain lacking intact nef genes [30–33]. A similar situation was reported in the French ALT cohort of long-term nonprogressor patients . Interestingly, expression of NKp44L was observed on HIV-infected cells from two out of six analyzed patients, presented profound alterations of nef sequences. However, in those patients, NKp44L is practically not present on noninfected cells, following PHA-activation, suggesting that this subset was highly sensitive to NK lysis, as compared with p24+ HIV-infected cells. The conserved ability of patient-derived Nef proteins to act on NKp44L cell-surface expression suggests that this activity is conserved in vivo. Of note, specific deletion observed in the cohort of Sydney was never observed in the LTNP individual of the ALT cohort . We cannot exclude a peculiar role of NK cells from these long-term nonprogressor patients; indeed, O'Connor et al.  have recently shown that these cells are characterized by altered phenotype and function including increased level of NK cytotoxicity, as compared to viremic HIV+ patients.
Nef sequestration of NKp44L within infected CD4+ T cells seems to be independent of the AP-1-dependent endocytotic pathway, the proline rich motif (PxxP), and others Nef domains previously described, including those implicated in the CD4 cell and HLA class-I downmodulation pathways. This suggests that NKp44L downmodulation capacity differs from previously mentioned activities of Nef. Interestingly, the mechanism of Nef downmodulation of CD80 cell and CD86 cell is also distinct from the one used for HLA class-I relocation . However, a common prerequisite to all Nef actions consists in its N-terminal myristoylation. For example, HIV-1 Nef upregulated CCL2/MCP1 expression in astrocytes in a myristoylation-dependent manner . The N-terminal region of Nef containing the myristoylation site has been shown to be crucial for the development of an AIDS-like disease in mice . N-myristoylation is a common lipid modification of proteins, which is essential for the function of proteins, involved in many cellular pathways of signal transduction and apoptosis. Modification of viral proteins by myristic acid plays an important role in different stages of the virus life cycle . The importance of Nef N-terminal myristoyl moiety for the inhibition of NKp44L cell-surface expression was confirmed by applying a myristoylation-deficient Nef variant. Furthermore, in presence of 2 OHM and 4 OXA, two specific myristoylation inhibitors, the efficiency of Nef to thwart the cell-surface expression of NKp44L and NK lysis was sharply decreased and strongly inhibited, respectively. Interestingly, in presence of these specific inhibitors a significant fraction of Nef was localized in the nucleus (data not shown), according to previous data which shown that in absence of myristoylation, Nef was more diffuse within the cytoplasm of infected cells, and it was also present in the nucleus of infected cells . Of note, the presence of Nef has no effect on the intracellular level of NKp44L (data not shown), strengthened the idea that Nef acts on the translocation pathway of NKp44L or induces the internalization of NKp44L presents at the cell surface. This suggests that the localization, or the integrity, or both, of Nef is a major point to maintain the down-modulation of NKp44L expression at the cell-surface level, and could also partially explain the controller status of a few particular patients.
Cerboni et al.  have recently shown that Nef also downmodulates cell-surface expression of MICA, ULBP1 and ULBP2, the cellular ligands of NKG2D, another activating receptor expressed by NK cells. The possibility that Nef may affect the expression of other activating NK receptors cannot be excluded.
In conclusion, by interfering with NKp44L, Nef likely helps HIV-1 to evade the host NK cell-mediated immune responses. This may facilitates the establishment of a permanent chronic infection and promotes the presence of a viral reservoir. This investigation brings key arguments to explain the paradoxal effect of HIV-1 infection, which induces in parallel the destruction of noninfected cells over-expressing NKp44L, and protects infected cells by downmodulation of NKp44L. Our findings also strengthen the key role of NK function against HIV attack and highlight the need for new therapeutic strategies to eradicate the HIV reservoir through the inhibition of Nef.
We thank S. Benichou (Institut Cochin, Paris, France) for providing Nef-GFP plasmid.
Sponsorship: This work was supported by grant from Agence Nationale de Recherche sur le SIDA (ANRS). H.F.B. was supported by a doctoral fellowship from the ANRS.
Author contributions: V.V., O.S., H.F-B. and P.D. conceived and designed the experiments; H.F-B. and V.V. performed research; V.V., O.S., N.S-F. and P.D. analyzed data; D.C. and H.A. enrolled patients from ALT cohort, and performed Nef sequencing; and V.V., O.S, H.F-B. and P.D. wrote the paper.
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