Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: J Infect Dis. 2014 Feb 14;210(3):410–418. doi: 10.1093/infdis/jiu094

Influenza virus uses its neuraminidase protein to evade the recognition of two activating NK cell receptors

Yotam Bar-On 1, Einat Seidel 1, Pinchas Tsukerman 1, Michal Mandelboim 2, Ofer Mandelboim 1
PMCID: PMC4074429  EMSID: EMS57377  PMID: 24532603

Abstract

Natural Killer (NK) cells play a central role in the defense against viral infections and in the elimination of transformed cells. The recognition of pathogen-infected and tumor cells is controlled by inhibitory and activating receptors. We have previously shown that among the activating (killer) NK cell receptors the Natural Cytotoxicity Receptors, NKp44 and NKp46, interact with the viral hemagglutinin (HA) protein expressed on the cell surface of influenza-virus-infected cells. We further showed that the interaction between NKp44/NKp46 and viral HA is sialic-acid dependent and that the recognition of HA by NKp44 and NKp46 leads to the elimination of the infected cells. Here we demonstrate that the influenza virus developed a counter-attack mechanism and that the virus uses its neuraminidase (NA) protein to prevent the recognition of HA by both the NKp44 and NKp46 receptors, resulting in reduced elimination of the infected cells by NK cells.

Introduction

The activity of NK cells is controlled by inhibitory signals derived from binding of NK inhibitory receptors to self ligands such as MHC class I, CEACAM1, PVR and phosphatidylethanolamine (PE) [1-6] and by activating signals derived from the binding of the NK activating receptors to viral proteins, tumor proteins, stress-induced ligands and even self ligands [5]. NK cells express several killer receptors, among which are the family of Natural Cytotoxicity Receptors (NCRs) which contain three members: two (NKp30 and NKp46) that are constitutively expressed and one (NKp44) whose expression is up-regulated upon NK cell activation [7-9]. Interestingly, mice express only one of these NCRs, the NKp46 orthologue protein Ncr1 [10, 11].

NKp44 was shown to be involved in many key NK-mediated functions such as tumor immune surveillance [12, 13], production of cytokines and growth factors by decidual NK cells [14] and controlling viral infection [15-17]. Two tumor cell ligands were identified for NKp44: the proliferating cell nuclear antigen (PCNA) that surprisingly inhibits NKp44 activity [18] and the mixed-lineage leukemia-5 (MLL5) that activates it [19]. Interestingly, both NKp44 ligands were reported to be expressed under normal conditions primarily in the nucleolus and in the cytoplasm, and it is still unclear how they get to the cell surface of tumor cells. In contrast, the interaction of NKp44 with several viruses is well characterized. Specifically, it was shown that NKp44 can activate NK cells against the influenza virus [16, 20] and against the new castle disease virus [17] by binding to their HA proteins. It was also demonstrated that NKp44 recognizes cells infected with Kaposi’s Sarcoma Herpesvirus (KSHV) [21], Dengue virus [22], HIV [23] and West Nile virus [22]. However, in all of these later cases, the molecular mechanisms by which NKp44 recognizes KSHV, dengue, HIV and West Nile virus are still largely unknown.

NKp44 cooperates with the other NCRs receptors, NKp46 and NKp30, to induce NK-mediated cytotoxicity against various target cells [24]. In addition, both NKp44 and NKp46 and the mouse Ncr1 recognize HA on influenza-virus-infected cells [25-30]. The binding of NKp44, NKp46 and Ncr1 to viral HA, which is mediated by specific sialic acids residues found on these receptors, leads to the elimination of the infected cells [26]. In the absence of Ncr1, enhanced sensitivity to influenza virus infection is observed [27]. The other NCR, NKp30, does not bind the HA of influenza virus and therefore does not contribute to the NK-mediated killing of influenza-virus-infected cells. However, this receptor was shown to bind the poxvirus HA and, surprisingly, this interaction inhibits the killing of poxvirus infected cells [31].

We have recently shown that the NA protein is also involved in NK cell recognition of infected cells, but in an opposite manner to that of HA. We demonstrated that the influenza virus utilizes the viral NA protein to evade the NKp46-mediated elimination and that inhibition of NA leads to increased elimination of influenza-virus-infected cells both in vitro and in vivo [32]. It is still unknown, however, whether NA antagonizes the activity of NKp44 and whether the NA-mediated neutralization of the recognition of NKp44 is important for the evasion of influenza from NK-cell-mediated elimination.

Materials and Methods

Cells, viruses and viral infection

The cell lines used in this study were the human choriocarcinoma cell line Jeg3, the mouse lymphoma cell line EL4 and the murine thymoma BW cell line. The human influenza virus A/Puerto Rico/8/34 H1N1 used in this study was generated as previously described [33].

Antibodies, fusion proteins and compounds

The monoclonal antibodies (mAbs) used in the present study included the anti-Influenza type A monoclonal mAb (Centers for Disease Control Atlanta Georgia), anti-influenza virus type A (H1) mAb (H17-L2) (the kind gift of Dr. Jonathan Yewdell, NIH), APC conjugated anti-human NKp44 mAb (BioLegend), LEAF Purified anti-human CD336 (NKp44), PE- conjugated anti-human NKp46 (Beckman Coulter) and PE-conjugated anti mouse Ncr1 (R&D systems). Biotin-SP-AffiniPure Rabbit Anti-Human IgG and anti human Fcγ polyclonal antibodies were purchased from Jackson ImmunoResearch. NKp44-Ig, NKp46-Ig, Ncr1-Ig, D1-Ig, KIR2DS4-Ig, KIR2DL1-Ig and HA-Ig fusion proteins were generated in the human embryonic kidney cells 293T and were purified on a protein G column as described [26]. For NA inhibition, oseltamivir carboxylate (Santa cruz, sc-212484) and zanamivir (Santa cruz, sc-208495) were used.

Western blot

NKp44-Ig, NKp46-Ig, Ncr1-Ig, NKp30-Ig, KIR2DL1-Ig and KIR2DS4-Ig (2μg) were run on 10% SDS-PAGE gels in reducing conditions and blotted with 0.4μg/ml of biotinylated HA-Ig or with biotinylated anti-human IgG (0.1μg/ml) and then incubated with Avidin-HRP (Bio Legend). For NA treatment, NKp44-Ig and NKp46-Ig fusion protein were incubated with NA beads (Sigma) at a ratio of 3.5μl NA beads for 1μg fusion protein and were treated or not with 25ul oseltamivir carboxylate (10ug-10mg/ml). Samples (4μg) were run on 10% SDS-PAGE gels and blotted with 20 μg/ml of biotinylated SNA lectin (Vector laboratories) or with 0.4μg/ml of biotinylated HA-Ig and then incubated for 30 minutes with Avidin-HRP (Bio Legend).

FACS staining of infected cells

For FACS staining of the infected cells, cells were incubated overnight with A/Puerto Rico/8/34 (H1N1) virus strain at 37°C. The cells were then washed and incubated with the appropriate fusion protein (5μg/well), with the influenza type A monoclonal mAb or with anti-influenza virus type A (H1) mAb (H17-L2) for 2 hr on ice, washed and then stained with the appropriate secondary labeled antibody. For NA inhibition assays, the cells were incubated overnight with A/Puerto Rico/8/34 (H1N1) virus strain at 37°C, washed, incubated with 10μg/ml of the indicated NA inhibitors for 1 hr on ice and then stained with the appropriate fusion proteins or mAb. BW cells were stained with APC-conjugated anti-human NKp44 mAb (BioLegend), PE-conjugated anti-human NKp46 (Beckman Coulter) and PE-conjugated anti mouse Ncr1 (R&D systems) for the detection of the relevant chimeric protein. All staining was analyzed by FACS using the CellQuest software.

BW assay

The BW transfected cells were prepared as previously described [33]. Fifty thousand BW or BW-transfected cells were incubated with the same amount of irradiated (6,000 rad) influenza-virus-infected EL4 cells for 8 h at 37 °C and 5% CO2. For NA inhibition, infected cells were incubates with 25μg/50,000 cells of NA inhibitor for 1hr on ice. Following 8 hr incubation, supernatants were collected and the level of IL-2 was quantified by ELISA using anti-IL-2 mAb. IL-2 secretion from transfected BW cells was normalized by reducing the background IL2 secretion of parental BW cells. Student’s t test and was used to determine significant differences.

Cytotoxicity assay and NK cell preparation

The cytotoxic activity of NK cells against various targets was assessed in 5 hr 35S release assays as described [34]. For NA inhibition, target cells were incubated with 25μg/well of the NA inhibitor for 1hr on ice. For blocking NKp44 and NKp46 activity, NK cells were incubated on ice for 1 hr with 0.5μg/well of anti-human CD336 (NKp44) antibody (BioLegend) or with 10ul/well of anti-human NKp46 (Beckman Coulter) prior to the incubation with the targets. The effector-to-target ratio was 15:1. Human NK cells were isolated from peripheral blood using the Human NK Cell Isolation Kit and the autoMACS instrument (Miltenyi Biotec) according to the manufacturer’s instructions and were grown as previously described [34]. Student’s t test and was used to determine significant differences.

Results

NKp44, NKp46 and Ncr1 directly interact with viral HA

We demonstrated previously that the activating receptors NKp44 and NKp46 recognize viral HA on infected cells and that this interaction (which is dependent on the sialylation of the receptors) leads to the killing of the infected cells by NK cells [25, 26, 28]. Furthermore, we showed that the mouse homologous protein of NKp46 called Ncr1 also binds to viral HA [27, 30]. To demonstrate that Ncr1, NKp44 and NKp46 directly interact with viral HA, we prepared fusion proteins composed of the extracellular portions of Ncr1, NKp44, NKp46 and of control NK cell receptors such as NKp30, KIR2DS4 and KIR2DL1 fused to the Fc portion of human IgG1. The proteins were purified on protein G columns (purity was around 95%) and ran on SDS-PAGE gels (Figure 1A). The SDS-PAGE gels were blotted with biotynylated HA-Ig (composed of the extracellular portion of the HA of the influenza virus A/Puerto Rico/8/34 H1N1 fused to the human IgG1 Fc domain (Figure 1A) and were also blotted with anti-human Fc antibody (Figure 1A) that was used as control. Importantly, direct recognition by HA-Ig of NKp46-Ig, Ncr1-Ig and NKp44-Ig but not of NKp30-Ig, KIR2DS4 and KIR2DL1 was observed (Figure 1A). The ratio between the biotynylated HA-Ig staining and the anti-human Fc antibody staining of each fusion protein is summaries in figure 1B.

Figure 1. NKp44, NKp46 and Ncr1 directly interact with the viral HA protein.

Figure 1

Open in a new tab

(A) The fusion proteins Ncr1-Ig, NKp46-Ig, NKp44-Ig, NKp30-Ig, KIR2DS4-Ig and KIR2DL1-Ig were run on SDS-PAGE gels and western blotted with biotinylated HA-Ig and with anti-human IgG1 Fc antibody. The Y-axis shows the quantification (in arbitrary units, A.U.) of the relative intensity of the HA-Ig binding (grey columns) or the anti-human IgG1 Fc antibody binding (black columns). Representative results from three independent experiments are shown. (B) A table summarsing of the ratio between the biotynylated HA-Ig staining and the anti-human Fc antibody staining of each fusion protein depict in figure 1A

The direct interaction of the activating receptors NKp44 and NKp46 with HA is impaired by viral NA

Viral HA binds terminal N-acetyl neuraminic acid residues (sialic acids) attached to galactose [26, 29]. NKp46, Ncr1 and NKp44 are all sialylated proteins that utilize this property to bind the viral HA protein on the infected cells [26, 29, 30]. To test whether the direct interaction between NKp44-Ig, NKp46-Ig and the HA protein observed above (Figure 1) is sialic-acid dependent we treated both NKp46-Ig and NKp44-Ig with NA and western blotted the treated and untreated proteins with SNA lectin (that binds specifically to Neu5NAcα(2,6) sialic acid residues) and with HA-Ig. As can be seen and as we previously published [32], NA treatment of NKp46 resulted in a reduction in the sialic acids content (Figure 2A, detected by the SNA lectin), which consequently lead to impaired HA-Ig binding (Figure 2B). Importantly, NA treatment of NKp44-Ig also lead to reduced sialic acids content and a complete abolishment of the SNA lectin binding was observed after overnight incubation with NA (Figure 2C). Consequently, abolishment of HA-Ig recognition was also observed (Figure 2D). The extended incubation of NKp44-Ig at 37 degrees either alone (Figure 2E), or with NA (data not shown) did not affect protein integrity. Collectively this indicates that the direct binding of NKp46 and NKp44 to viral HA is sialic-acid dependent and that similar to NKp46 binding, the direct NKp44-binding to viral HA is also impaired by the viral NA.

Figure 2. The direct binding of NKp44 and NKp46 to HA is reduced following NA treatment.

Figure 2

Open in a new tab

NKp46-Ig (A and B) and NKp44-Ig (C, D and E) were incubated with and without NA beads (the NKp44-Ig was incubated for various periods of time, indicated in the x axis of C, D and E), run on SDS-PAGE gels and then western blotted with SNA lectin (A and C), with biotinylated HA-Ig (B and D) and with anti-human Fc antibody (E). The graphs above the western blots show the quantification (in arbitrary units, A.U.) of the relative intensity of the SNA lectin, the HA-Ig and anti-human Fc antibody binding. The present or absence of NA beads is indicated below the western blot gels. Representative results from three independent experiments are shown.

Enhanced binding of all HA-interacting proteins to influenza-virus-infected cells following NA inhibition

Since we demonstrated above (Figure 2), and previously regarding NKp46/Ncr1 [32], that NA impairs the HA recognition by NKp44, we next tested whether inhibition of NA leads to increased NKp44-Ig, Ncr1-Ig and NKp46-Ig recognition of infected cells. To address this issue, influenza-virus-infected Jeg3 cells were stained with NKp44-Ig, NKp46-Ig, Ncr1-Ig and with a truncated NKp46 fusion protein (D1-Ig) lacking the HA-interacting domain [26], serving therefore as a negative control. Staining of the infected cells was performed with and without NA inhibition, which was mediated by osetamivir carboxylate (O. carboxylate) and zanamivir. In the absence of influenza, little or no binding was observed (data not shown). Upon infection, increased binding of NKp44-Ig, NKp46-Ig and Ncr1-Ig was seen (Figure 3A). Importantly, when NA was inhibited with O. carboxylate (Figure 3B) or with zanamivir (Figure 3C), increased binding of NKp46-Ig, Ncr1-Ig and NKp44-Ig to the influenza-virus-infected cells was observed. No D1-Ig binding was seen prior to and following the treatments (Figure 3A-C). To further control these experiments, we stained both treated and untreated influenza-virus-infected cells with an anti-influenza virus type A HA mAb and with anti-influenza type A mAb and observed that the untreated and treated cells were similarly recognized by both mAbs (Figure 3D). Altogether, this indicates that inhibiting the NA activity leads to a significant increase in the binding of Ncr1, NKp46 and NKp44 to influenza-virus-infected cells.

Figure 3. The binding of NKp44-Ig, NKp46-Ig and Ncr1-Ig to influenza-virus-infected cells is enhanced following the blocking of NA Activity.

Figure 3

Open in a new tab

(A-D) FACS staining of influenza-virus-infected Jeg3 cells with D1-Ig, NKp44-Ig, NKp46-Ig and Ncr1-Ig prior (A) and following NA inhibition with O. carboxylate (B) or with zanamivir (C). In each panel the empty black histograms depict staining with the indicated fusion protein and the filled gray histogram depicts the staining with the secondary mAb only. (D) FACS analysis of infected cells stained with anti-HA mAb (left histogram) and with anti-influenza type A mAb (right histogram). The empty black histogram depicts the binding of the untreated cells and the empty dashed gray histogram depicts the staining of O. carboxylate treated cells. The filled gray histogram depicts the staining with the secondary mAb only. In all figure parts the staining is presented after gating of the live-cell population only. The figure shows representative staining. Three independent experiments were performed.

We also tested whether influenza infection of mouse cells would result in increased Ncr1, NKp44 and NKp46 binding to the infected cells following NA treatment. In agreement with the results obtained with human cells, following NA inhibition with O. carboxylate, increased NKp44-Ig, NKp46-Ig and Ncr1-Ig binding to EL4-infected cells was observed, but no change in anti-HA mAb binding was detected (Figure 4).

Figure 4. The binding of NKp44-Ig, NKp46-Ig and Ncr1-Ig to influenza-virus-infected EL-4 cells is enhanced following NA inhibition.

Figure 4

Open in a new tab

EL4 cells were infected with influenza virus and then stained with anti-HA mAb, NKp44-Ig, Ncr1-Ig and NKp46-Ig (all indicated in the x axis) prior to (empty black graph) and following (empty gray graph) NA inhibition with O. carboxylate. The filled gray histogram depicts the staining with the secondary mAb only. In all figure parts the staining is presented after gating of the live cell population only. The figure shows representative staining. Three independent experiments were performed.

Increased reporter cell activity following NA inhibition

We next tested whether the inhibition of NA activity that leads to increased Ncr1, NKp46 and NKp44 recognition of infected cells would result in increased function of these receptors. For that we initially used a cell-based reporter system in which we expressed in-mouse BW cells chimeric proteins composed of the extra cellular portion of NKp44, Ncr1 or NKp46 proteins fused to the mouse zeta chain (BW NKp44, BW Ncr1 and BW NKp46, respectively). In this cell-based reporter assay, the BW cells secrete IL-2 upon engagement of the chimeric protein with an appropriate antigen/ligand [1]. After verifying the expression of the chimeric proteins (Figure 5A), the various BW cells were incubated for 8 hrs with influenza-virus-infected EL-4 cells that were treated or not with O. carboxyalte. Following this incubation period, IL-2 in the supernatants was determined by ELISA. In accordance with the increased NKp44 and NKp46/Ncr1 binding observed following O. carboxyalte treatment of the influenza-virus-infected cells (Figure 4), increased IL-2 secretion from the various BW cells was also detected following the O. carboxyalte treatment (Figure 5B). No increase in IL-2 secretion by parental BW cells was seen following NA inhibition (Figure 5B).

Figure 5. NA Inhibition leads to increased activity of reporter cell lines.

Figure 5

Open in a new tab

(A) FACS staining of BW cells expressing the NKp44-zeta (BW NKp44), Ncr1-Zeta (BW Ncr1) and NKp46-Zeta (BW NKp46) chimeric proteins. In each panel the empty black histogram depicts the staining with the indicated mAb, and the filled gray histogram depicts the staining of parental BW cells with the indicated mAb. (B) The various BW cells expressing the chimeric proteins seen in (A) were incubated with influenza-virus-infected EL-4 cells that were treated or not with O. carboxyalte. IL-2 secretion was determined by ELISA 8 hrs following the incubation with the infected cells. Shown is the fold increase of IL-2 secretion (treated cells/untreated cells). The mean values and SD derived from triplicates. Statistically significant differences are indicated (*p < 0.01, **p < 0.001).

Blocking of NA resulted in increased NKp44 and NKp46-dependent killing

In our final set of experiments we wanted to determine whether NA impairs the activity of both NKp46 and NKp44. We performed NK cytotoxicity assays using the Jeg3 cell line and human NK cells. In the absence of infection, only minimal killing of Jeg3 cells was observed (since Jeg3 do not express known ligands for NK cells [32]), and upon influenza infection, NK killing was observed (Figure 6). Importantly, when infected cells were pretreated with O. carboxylate, a significant increase in NK cytotoxicity was observed, and this increased killing was reduced when either NKp44 or NKp46 receptors were blocked (Figure 6). Moreover, when both NKp44 and NKp46 receptors were blocked together, the increased NK-mediated killing was significantly reduced in comparison to blocking only one of these receptors (Figure 6). These results demonstrate that the triggering of NKp44 or NKp46 killer receptors by HA can by itself induce NK-mediated killing of infected cells, that NA impairs the activity of both NKp44 and NKp46, and that blocking of NA activity results in increased elimination of the infected cells.

Figure 6. Inhibition of NA activity boosts NKp44 and NKp46 killing.

Figure 6

Open in a new tab

(A) Uninfected and influenza-infected Jeg3 cells were tested in killing assays in the presence or in the absence of O. carboxyalte, with or without anti-NKp44 and anti-NKp46 blocking mAb (the various treatments are indicated in the x axis). The effector-to-target ratio (E:T) was 15:1. Shown are mean values and SD derived from triplicates. Statistically significant differences are indicated (*p < 0.05, **p<0.05, one-tailed student’s t-test). The figure shows the results of one representative experiment out of three performed

Discussion

NK cells have developed multiple strategies to recognize and eliminate virus infected cells. The significance of this NK activity is illustrated by the variety of mechanisms that viruses have developed to evade the NK cell recognition. For example, human cytomegalovirus (HCMV), in order to achieve long and persistent infection in the host, uses different mechanisms that include viral proteins and miRNAs to evade the NKG2D activating receptor recognition [35-39].

The activity of the NCRs NKp30, NKp44 and NKp46 is also manipulated by different viruses [21, 31, 33]. For example, it was shown that the viral tegument protein pp65 binds to NKp30 and inhibits its activity [40]. For NKp44 it was demonstrated that KSHV uses the ORF54/dUTPase protein to down regulate the expression of an unknown NKp44 ligand, expressed on infected cells [21]. For NKp46/Ncr1, we have recently demonstrated that the viral NA protein of influenza virus antagonizes its activity [32].

NKp44 and NKp46/Ncr1 are the major NK receptors responsible for the detection and elimination of influenza-virus-infected cells [26]. This is mediated by the binding of these activating receptors to the viral HA protein on the membrane of the infected cells or on cells that are coated with the virus [26, 32]. HA is a central protein in the virus’s life cycle, as its property to bind sialic acids on the membrane of the target cells enables the infection of these cells [41]. The ability of HA to bind sialic acids residues is conserved among all influenza viruses, and indeed although approximately ten thousand different sequences of HA proteins are found in the data bank, they all probably share this common feature of sialic-acid recognition. We suggest that NK cells utilize this general HA property to bind sialic acids in order to recognize the infected cells via NKp46 and NKp44.

Influenza viruses, on the other hand, did not remain passive, and since the sialic acid binding property of the virus could not be changed, the virus developed other strategies to evade the NK-cell recognition. As we have recently shown, influenza virus uses the sialidase activity of the viral NA to remove sialic acids from the NKp46 receptor and its mouse orthlogue Ncr1 protein in order to impair the recognition of infected cells [32]. Here we demonstrate that NA impairs the activity of NKp44 as well.

To test whether the NA-mediated immune evasion mechanism is NKp46 specific or whether it can be used by the influenza virus to evade other NK activating receptors, we first demonstrated that NKp46/Ncr1 and NKp44 directly interact with viral HA. The direct binding between the NKp46/Ncr1, NKp44 and the viral HA was seen in western blot assays under denaturative conditions. This further emphasizes that the binding between NKp46, Ncr1, NKp44 and viral HA is largely dependent on the sialic acids recognition and that conformational protein epitopes are probably less important for this recognition. Nevertheless, a certain degree of specificity is observed because other sialylated proteins such as NKp30, KIR2DL1 and KIR2DS4 do not interact with viral HA. Indeed, for NKp46 we demonstrated that the sialylated residue Threonine, located in position 225, and not the other two glycosylated residues of NKp46, is primarily responsible for the NKp46 interaction with viral HA [26]. The exact binding sites through which Ncr1 and NKp44 binds to HA are still unknown.

Our data indicates that the influenza virus uses its NA protein not only to evade the recognition of NKp46 but also to broadly prevent the activation of NK cells, and that this is achieved by impairing the recognition of NKp44 in addition to NKp46/Ncr1 [32]. We further demonstrate that both killer proteins NKp44 and NKp46 are important in the elimination of influenza-virus-infected cells and that blocking the activity of both receptors significantly reduces the killing of infected cells.

The realization that NA inhibitors not only block virus infection and budding but also boost the NKp44 and NKp46 recognition might lead to the development of new drugs that utilize the activity of NKp44 and NKp46 to prevent or treat influenza virus infection.

Acknowledgments

We thank J. Yewdell for providing the anti-HA antibody.

Funding This study was supported by the Advanced ERC grant, The Israeli Science Foundation, The Israeli- I-CORE, the GIF foundation and by The ICRF professorship grant (all to O.M). O.M is a Crown professor of Molecular Immunology.

Footnotes

The authors have no conflict of interest.

References

  • 1.Stanietsky N, Simic H, Arapovic J, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci U S A. 2009;106:17858–63. doi: 10.1073/pnas.0903474106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Simhadri VR, Andersen JF, Calvo E, Choi SC, Coligan JE, Borrego F. Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells. Blood. 2012;119:2799–809. doi: 10.1182/blood-2011-08-372425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Long EO, Kim HS, Liu D, Peterson ME, Rajagopalan S. Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol. 2013;31:227–58. doi: 10.1146/annurev-immunol-020711-075005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hosomi S, Chen Z, Baker K, et al. CEACAM1 on activated NK cells inhibits NKG2D-mediated cytolytic function and signaling. Eur J Immunol. 2013 doi: 10.1002/eji.201242676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kirwan SE, Burshtyn DN. Regulation of natural killer cell activity. Curr Opin Immunol. 2007;19:46–54. doi: 10.1016/j.coi.2006.11.012. [DOI] [PubMed] [Google Scholar]
  • 6.Lankry D, Rovis TL, Jonjic S, Mandelboim O. The interaction between CD300a and phosphatidylserine inhibits tumor cell killing by NK cells. Eur J Immunol. 2013;43:2151–61. doi: 10.1002/eji.201343433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Moretta A, Bottino C, Vitale M, et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol. 2001;19:197–223. doi: 10.1146/annurev.immunol.19.1.197. [DOI] [PubMed] [Google Scholar]
  • 8.Cantoni C, Ponassi M, Biassoni R, et al. The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity. Structure. 2003;11:725–34. doi: 10.1016/s0969-2126(03)00095-9. [DOI] [PubMed] [Google Scholar]
  • 9.Seidel E, Glasner A, Mandelboim O. Virus-mediated inhibition of natural cytotoxicity receptor recognition. CMLS. 2012;69:3911–20. doi: 10.1007/s00018-012-1001-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gazit R, Gruda R, Elboim M, et al. Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1. Nat Immunol. 2006;7:517–23. doi: 10.1038/ni1322. [DOI] [PubMed] [Google Scholar]
  • 11.Biassoni R, Pessino A, Bottino C, Pende D, Moretta L, Moretta A. The murine homologue of the human NKp46, a triggering receptor involved in the induction of natural cytotoxicity. Eur J Immunol. 1999;29:1014–20. doi: 10.1002/(SICI)1521-4141(199903)29:03<1014::AID-IMMU1014>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  • 12.Marras F, Bozzano F, De Maria A. Involvement of activating NK cell receptors and their modulation in pathogen immunity. BioMed Research International. 2011:2011:152430. doi: 10.1155/2011/152430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cantoni C, Bottino C, Vitale M, et al. NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J Exp Med. 1999;189:787–96. doi: 10.1084/jem.189.5.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hanna J, Goldman-Wohl D, Hamani Y, et al. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med. 2006;12:1065–74. doi: 10.1038/nm1452. [DOI] [PubMed] [Google Scholar]
  • 15.Ho JW, Hershkovitz O, Peiris M, et al. H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44. J Virol. 2008;82:2028–32. doi: 10.1128/JVI.02065-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Arnon TI, Lev M, Katz G, Chernobrov Y, Porgador A, Mandelboim O. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur J Immunol. 2001;31:2680–9. doi: 10.1002/1521-4141(200109)31:9<2680::aid-immu2680>3.0.co;2-a. [DOI] [PubMed] [Google Scholar]
  • 17.Jarahian M, Watzl C, Fournier P, et al. Activation of natural killer cells by newcastle disease virus hemagglutinin-neuraminidase. J Virol. 2009;83:8108–21. doi: 10.1128/JVI.00211-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rosental B, Brusilovsky M, Hadad U, et al. Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44. J Immunol. 2011;187:5693–702. doi: 10.4049/jimmunol.1102267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Baychelier F, Sennepin A, Ermonval M, Dorgham K, Debre P, Vieillard V. Identification of a cellular ligand for the natural cytotoxicity receptor NKp44. Blood. 2013;122:2935–42. doi: 10.1182/blood-2013-03-489054. [DOI] [PubMed] [Google Scholar]
  • 20.Arnon TI, Markel G, Mandelboim O. Tumor and viral recognition by natural killer cells receptors. Semin Cancer Biol. 2006;16:348–58. doi: 10.1016/j.semcancer.2006.07.005. [DOI] [PubMed] [Google Scholar]
  • 21.Madrid AS, Ganem D. Kaposi’s sarcoma-associated herpesvirus ORF54/dUTPase downregulates a ligand for the NK activating receptor NKp44. J Virol. 2012;86:8693–704. doi: 10.1128/JVI.00252-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hershkovitz O, Rosental B, Rosenberg LA, et al. NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells. J Immunol. 2009;183:2610–21. doi: 10.4049/jimmunol.0802806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Vieillard V, Strominger JL, Debre P. NK cytotoxicity against CD4+ T cells during HIV-1 infection: a gp41 peptide induces the expression of an NKp44 ligand. Proc Natl Acad Sci U S A. 2005;102:10981–6. doi: 10.1073/pnas.0504315102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pende D, Parolini S, Pessino A, et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp Med. 1999;190:1505–16. doi: 10.1084/jem.190.10.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mandelboim O, Lieberman N, Lev M, et al. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature. 2001;409:1055–60. doi: 10.1038/35059110. [DOI] [PubMed] [Google Scholar]
  • 26.Arnon TI, Achdout H, Lieberman N, et al. The mechanisms controlling the recognition of tumor- and virus-infected cells by NKp46. Blood. 2004;103:664–72. doi: 10.1182/blood-2003-05-1716. [DOI] [PubMed] [Google Scholar]
  • 27.Gazit R, Gruda R, Elboim M, et al. Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1. Nat Immunol. 2006;7:517–23. doi: 10.1038/ni1322. [DOI] [PubMed] [Google Scholar]
  • 28.Achdout H, Meningher T, Hirsh S, et al. Killing of avian and Swine influenza virus by natural killer cells. J virol. 2010;84:3993–4001. doi: 10.1128/JVI.02289-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mendelson M, Tekoah Y, Zilka A, et al. NKp46 O-glycan sequences that are involved in the interaction with hemagglutinin type 1 of influenza virus. J virol. 2010;84:3789–97. doi: 10.1128/JVI.01815-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Glasner A, Zurunic A, Meningher T, et al. Elucidating the mechanisms of influenza virus recognition by Ncr1. PLoS One. 2012;7:e36837. doi: 10.1371/journal.pone.0036837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Jarahian M, Fiedler M, Cohnen A, et al. Modulation of NKp30- and NKp46-mediated natural killer cell responses by poxviral hemagglutinin. PLoS Pathog. 2011;7:e1002195. doi: 10.1371/journal.ppat.1002195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Bar-On Y, Glasner A, Meningher T, et al. Neuraminidase-mediated, NKp46-dependent immune-evasion mechanism of influenza viruses. Cell Rep. 2013;3:1044–50. doi: 10.1016/j.celrep.2013.03.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Achdout H, Arnon TI, Markel G, et al. Enhanced recognition of human NK receptors after influenza virus infection. J Immunol. 2003;171:915–23. doi: 10.4049/jimmunol.171.2.915. [DOI] [PubMed] [Google Scholar]
  • 34.Mandelboim O, Reyburn HT, Vales-Gomez M, et al. Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules. J Exp Med. 1996;184:913–22. doi: 10.1084/jem.184.3.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Stern-Ginossar N, Elefant N, Zimmermann A, et al. Host immune system gene targeting by a viral miRNA. Science. 2007;317:376–81. doi: 10.1126/science.1140956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Nachmani D, Stern-Ginossar N, Sarid R, Mandelboim O. Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells. Cell host microbe. 2009;5:376–85. doi: 10.1016/j.chom.2009.03.003. [DOI] [PubMed] [Google Scholar]
  • 37.Nachmani D, Lankry D, Wolf DG, Mandelboim O. The human cytomegalovirus microRNA miR-UL112 acts synergistically with a cellular microRNA to escape immune elimination. Nat Immunol. 2010;11:806–13. doi: 10.1038/ni.1916. [DOI] [PubMed] [Google Scholar]
  • 38.Fernandez-Messina L, Reyburn HT, Vales-Gomez M. Human NKG2D-ligands: cell biology strategies to ensure immune recognition. Front Immunol. 2012;3:299. doi: 10.3389/fimmu.2012.00299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wilkinson GW, Tomasec P, Stanton RJ, et al. Modulation of natural killer cells by human cytomegalovirus. Journal of clinical virology. 2008;41:206–12. doi: 10.1016/j.jcv.2007.10.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Arnon TI, Achdout H, Levi O, et al. Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus. Nat Immunol. 2005;6:515–23. doi: 10.1038/ni1190. [DOI] [PubMed] [Google Scholar]
  • 41.Nayak DP, Hui EK, Barman S. Assembly and budding of influenza virus. Virus research. 2004;106:147–65. doi: 10.1016/j.virusres.2004.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES