Elucidating the Mechanisms of Influenza Virus Recognition by Ncr1

Natural killer (NK) cells are innate cytotoxic lymphocytes that specialize in the defense against viral infection and oncogenic transformation. Their action is tightly regulated by signals derived from inhibitory and activating receptors; the later include proteins such as the Natural Cytotoxicity Receptors (NCRs: NKp46, NKp44 and NKp30). Among the NCRs, NKp46 is the only receptor that has a mouse orthologue named Ncr1. NKp46/Ncr1 is also a unique marker expressed on NK and on Lymphoid tissue inducer (LTI) cells and it was implicated in the control of various viral infections, cancer and diabetes. We have previously shown that human NKp46 recognizes viral hemagglutinin (HA) in a sialic acid-dependent manner and that the O-glycosylation is essential for the NKp46 binding to viral HA. Here we studied the molecular interactions between Ncr1 and influenza viruses. We show that Ncr1 recognizes influenza virus in a sialic acid dependent manner and that N-glycosylation is important for this binding. Surprisingly we demonstrate that none of the predicted N-glycosilated residues of Ncr1 are essential for its binding to influenza virus and we thus conclude that other, yet unidentified N-glycosilated residues are responsible for its recognition. We have demonstrated that N glycosylation play little role in the recognition of mouse tumor cell lines and also showed the in-vivo importance of Ncr1 in the control of influenza virus infection by infecting C57BL/6 and BALB/c mice knockout for Ncr1 with influenza

Human NKp46 and mouse Ncr1 structure.

<p>The figure shows a schematic description of the human NKp46 (A) and mouse Ncr1 (B) domains and glycosylation positions. White circle: N-linked glycosylation; Grey circle: O-linked glycosylation.</p

N-glycosylation plays little role in tumor cell recognition.

<p>(<b>A</b>) FACS staining of various tumor cell lines (indicated above the histograms) with control fusion protein NKp46-D1-Ig (gray filled histograms) and with Ncr1-Ig (black line) generated in COS-7 cells (upper lane), generated in HEK293T cells (second lane up) and generated in HEK293T cells and mutated at the indicated positions (lower lanes). (<b>B</b>) FACS staining of various tumor cell lines (indicated above the histograms) with control fusion protein NKp46-D1-Ig (gray filled histograms) with WT (upper and third rows) and triple mutated (second and bottom rows) Ncr-Ig fusion proteins (black line) untreated (two upper rows) or treated with NA (two lower rows).</p

The increased binding of Ncr1-Ig to PR8-coated EL4 and PD1.6 cells is NA-sensitive.

<p>The figure shows FACS staining of uncoated (left), PR8 coated 10 µl, 1000 HU (center) and 20 µl, 1000 HU (right) EL4 (<b>A</b>) and PD1.6 (<b>B</b>). The various cells, untreated, (upper histograms), or NA treated (lower histograms) were stained with Ncr1-Ig (black line), and with a control fusion protein NKp46-D1-Ig (gray filled histograms) Fluorescent intensity (MFI) is presented as the ratio staining/background and is indicated on the upper right side of each histogram. The figure is representative of three independent experiments.</p

Ncr1 is critical for <i>in-vivo</i> influenza eradication.

<p>C57BL/6 (<b>A</b>) and BALB/c (<b>B</b>) WT and KO mice were infected with two doses of PR8 influenza, as indicated. Survival was assessed using the Kaplan Meier model and the Tarone-Ware test. Termination point of the experiment was set to 20 days and mice that survived the infection were then considered healthy. The figure represents one of two independent experiments; at least 7 mice were used in each group. *P<0.05, **P<0.005.</p

N-glycosylation is required for Ncr1-Ig binding to PR8-coated cells.

<p>The figure shows FACS staining of uncoated (<b>A</b>) and PR8 coated (20 µl,1000 HU, B-E) 721.221 cells. Staining was performed with Ncr1-Ig (black line), and a control fusion protein NKp46-D1-Ig (gray filled histograms). Coated cells were stained with Ncr1-Ig that underwent various treatments; untreated (<b>B</b>), treated with NA (<b>C</b>), treated with Protein Glycosidase F (PNGase F) (<b>D</b>), treated with a cocktail of O-deglycosylating enzymes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036837#s2" target="_blank">materials and methods</a>) (<b>E</b>). Fluorescent intensity (MFI) is presented as the ratio staining/background and is indicated on the upper right side of each histogram. The figure is representative of three independent experiments.</p

Ncr1 mediated killing.

<p>(<b>A</b>) FACS staining of PBLs with Ncr1 mAb. The figure shows the intrinsic GFP expression of the NK cells and is representative of two independent staining. (<b>B</b>) Line chart depicting 35S methionine Re-directed killing assay. Labeled p815 cells were pre-incubated with anti-Ncr1 mAb and then incubated with PBLs harvested 18 hours following 200 µg poly(I):poly(C) in 200 µl PBS administered by i.p. injection to WT and KO mice of the C57BL/6 and BALB/c strains. Values are shown as mean ±SEM. The figure is representative of two independent experiments. ***; P<0.0005, **; P<0.005 (<b>C</b>) line chart depicting 35S methionine killing assay. PBLs were harvested 18 hours following 200 µg poly(I):poly(C) in 200 µl PBS administered by i.p. injection to WT and KO mice of the C57BL/6 and BALB/c strains and incubated with labeled EL4 cells. At least 8 mice were used in each group; a representative of three independent experiments is shown. Values are shown as mean ±SEM P<0.05.</p