The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

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   FIG 1

   Expression of MGL1 on murine MΦ correlates with susceptibility to infection by IAV. (Ai) RAW 264.7 MΦ were cultured for 48 h with or without 400 ng/ml of recombinant murine IL-4, incubated with or without 5 mg/ml ASF for 30 min at 37°C, and infected with 10⁴ PFU of BJx109 for 1 h at 37°C. After washing, cells were incubated a further 7 h in the presence of ASF, fixed, stained for expression of newly synthesized viral NP, and examined by immunofluorescence. Data show the mean percent infection ± 1 standard deviation (SD) from 3 independent experiments. (Aii) RAW 264.7 MΦ were transfected with control (siScr) or MGL1-specific (siMGL) siRNA, cultured an additional 48 h, and infected with 10⁵ PFU of BJx109 in serum-free medium and cultured, fixed, and stained as described for panel Ai. Data from 1 of 2 independent experiments are shown. For panels i and ii, Student's t test was used for statistical analysis (***, P ≤ 0.001). (Bi) Western blot analysis confirming expression of MMR by PEC MΦ from C57BL/6 (B6) but not MMR^−/− mice. Proteins were resolved by SDS-PAGE under nonreducing conditions, transferred to PVDF membrane, and detected using MMR-specific MAb. Molecular weight markers (M) are shown. (Bii) Cell surface expression of MGL by PEC MΦ of C57BL/6 mice or MMR^−/− mice. Cells were stained with biotin-labeled MGL-specific MAb (solid histograms) or isotype control (dashed histograms). (Biii) Susceptibility of MΦ to infection by BJx109. Monolayers of PEC MΦ were pretreated with 10 mg/ml mannan (Mn) or 5 mg/ml ASF or were mock treated (−) and infected with 10⁶ PFU BJx109. Data are representative of 2 independent experiments. One-way ANOVA was used for statistical analysis (P ≤ 0.01 [**] and P ≤ 0.001 [***] compared to percent infection of the respective mock-treated samples). (C) Responses of MMR^−/− to IAV strains BJx109 (BJ) and PR8. PEC MΦ from MMR^−/− mice were infected with 10⁶ PFU of each virus. (Ci) At 8 h, cells were fixed, stained for expression of viral NP, and assessed by immunofluorescence. (Cii) At 24 h, cells were fixed and stained for nucleic acids using PI, and the number of adherent cells remaining was determined by immunofluorescence. Data from panels Ci and Cii are representative of 3 independent experiments and were analyzed using Student's t test (***, P ≤ 0.001). (Ciii) At 24 h, cell supernatants were clarified and assayed for IL-6, MCP-1, TNF-α, and MIP-2 as described in Materials and Methods. Detection limits of each assay are indicated by the dotted lines. One-way ANOVA was used for statistical analysis (P ≤ 0.01 [**] and P ≤ 0.001 compared to mock-treated samples).
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   FIG 2

   Lec1 cells are less susceptible to IAV infection than parental CHO cells. (A and B) CHO and Lec1 cells were incubated in medium alone (untreated; black histograms) or medium supplemented with 100 mU/ml of bacterial sialidase (sialidase; gray histograms), washed, and used in binding assays. (A) Binding of biotin-labeled MAA (i) or L-PHA (ii) was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone. MFI (± 1 SD) of binding to mock- or sialidase-treated cells for MAA (i) were the following: CHO, 31.2 ± 17.5 and 5.2 ± 3.6, respectively; Lec1, 35.6 ± 1.7 and 7.9 ± 1.4, respectively. MFI (± 1 SD) for L-PHA (ii) were the following: CHO, 13.6 ± 2.5 and 14.1 ± 1.4, respectively; Lec1, 1.4 ± 0.2 and 1.4 ± 0.2, respectively. (B) Binding of 5 μg/ml biotin-labeled BJx109 was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone. MFI (± 1 SD) for binding to mock- or sialidase-treated cells are shown (CHO cells, 395.3 ± 2.3 and 5.5 ± 0.1, respectively; Lec1 cells, 41.4 ± 4.2 and 3.7 ± 0.1, respectively.) (C and D) Monolayers of CHO or Lec1 cells were infected with 10⁷ PFU of BJx109 at 37°C for 1 h as described in Materials and Methods. At 2 or 8 h, cells were fixed and stained for expression of viral NP or HA. (C) Representative images of CHO cells (i) and Lec1 cells (ii) using a Leica DMLB microscope (used to determine the percentage of IAV-infected cells) are shown. Images show NP or HA (FITC) and double-stranded nucleic acid (DAPI). (D) Fluorescent and total cells were counted in at least 4 random fields (>200 cells) of CHO and Lec1 cells at 8 h postinfection. Data show the mean percent infection (± 1 SD) and are representative of 5 independent experiments. Student's t test was used to determine significance (***, P ≤ 0.001). (E) Flow cytometry to determine expression of IAV NP at 2 h (i) or 8 h (ii) after infection with BJx109. Representative histograms from IAV-infected (white histograms) or mock-infected (gray histograms) triplicate samples and mean percentages of infected cells (± 1 SD) are shown. A gate was set to include 5% of cells in uninfected controls, and the percentage of infected cells was determined relative to this. Data are representative of 3 independent experiments.
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   FIG 3

   MGL1 expressed by Lec1 cells retains calcium-dependent C-type lectin activity. (A) Cell surface expression of MGL1 (solid histograms) on Lec1-MGL1 (black histogram) and Lec1-ctrl cells (gray histogram) was determined by flow cytometry using an MGL-specific MAb. Unstained cells (un; dashed lines) are included for comparison. (B) Western blot analysis confirming expression of MGL in cell lysates from Lec1-MGL1 cells but not Lec-1-ctrl cells. Proteins were resolved by SDS-PAGE under nonreducing conditions, transferred to PVDF membrane, and detected using MGL-specific MAb. Data are representative of 3 independent experiments. (C) Binding of galactose-PAA to Lec1-MGL1 (black histogram) but not Lec1-ctrl cells (gray histogram) in buffer containing either 10 mM CaCl₂ (solid histograms) or 5 mM EDTA (dashed lines). Data are representative of 2 independent experiments.
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   FIG 4

   Expression of MGL1 by Lec1 cells results in enhanced IAV binding and increased susceptibility to IAV infection. (A) Binding of 5 μg/ml biotin-labeled BJx109 to CHO-ctrl (i), Lec1-ctrl (ii), and Lec1-MGL1 (iii) cells was determined by flow cytometry in the presence of 10 mM CaCl₂ (black histograms) or 0.5 mM EDTA (gray histograms). Representative histograms of triplicate samples are shown, and data were used to calculate mean MFI (± 1 SD) for each cell type in Ca²⁺ or in EDTA: CHO-ctrl, 389.7 ± 19.0 and 400 ± 28.6, respectively; Lec1-ctrl, 16.5 ± 3.2 and 13.5 ± 1.7, respectively; Lec1-MGL1, 77.2 ± 1.4 and 18.8 ± 6.4, respectively (P ≤ 0.001 [***] compared to Lec1-MGL1 in the presence of Ca²⁺; Student's t test). (B) Proteins in cell lysates of Lec1-MGL1 cells were resolved by SDS-PAGE under nonreducing conditions, transferred to PVDF membrane, and probed with purified BJx109 in VOPBA as described in Materials and Methods. Arrow indicates Ca²⁺-dependent binding to a species of ∼35 kDa, corresponding to the molecular mass of MGL1. (C) Lec1-ctrl or Lec1-MGL1 cells were infected with 10⁷ PFU of BJx109, and expression of newly synthesized viral NP (i) or HA (ii) was determined 8 h later. Data represent mean percent infection (± 1 SD) and are representative of 2 independent experiments (***, P ≤ 0.001; Student's t test). (D) Flow cytometry to determine expression of IAV NP (i) or HA (ii) at 8 h after infection with BJx109. Representative histograms from IAV-infected (white histograms) or mock-infected (gray histograms) cells are shown along with the mean percentage of infected cells (± 1 SD) from triplicate samples. A gate was set to include 5% of cells in uninfected controls, and the percentage of infected cells was determined relative to this. Data are representative of 2 independent experiments.
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   FIG 5

   Role of cell surface sialic acid and dynamin-dependent endocytosis in MGL1-mediated infection of Lec1 cells by IAV. (A) Monolayers of parental CHO-ctrl and Lec1-MGL1 cells were mock treated or treated with 100 mU/ml bacterial sialidase (Sial) for 60 min at 37°C, followed by incubation with 5 mg/ml ASF or medium alone at 4°C for 30 min. Cells were then incubated with 10⁷ PFU of BJx109 for 1 h at 4°C to allow virus binding, washed, and incubated for a further 7 h at 37°C. (B) Monolayers of parental CHO-ctrl and Lec1-MGL1 cells were incubated with 10⁷ PFU of BJx109 alone (mock) or in the presence of 50 μM dynasore for 1 h at 37°C, washed, and incubated for a further 7 h in the presence or absence of dynasore. Cells were fixed and stained for expression of newly synthesized viral NP as described in Materials and Methods. Cells were fixed, stained, and examined by immunofluorescence as described in the text. CHO-ctrl cells incubated with 10⁶ FFU of PIV-3 in the presence or absence of dynasore were fixed and stained at 16 h postinfection. Data represent the mean percent infection (± 1 SD) and are representative of 2 independent experiments. ***, significantly different from mock-treated control cells (P ≤ 0.001; one-way ANOVA); #, significantly different from mock-treated Lec1-MGL1 cells (P ≤ 0.001; one-way ANOVA).
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   FIG 6

   IAV strains differ in expression of galactose-rich glycans and in the ability to infect cells expressing MGL1. (A) Binding of plant lectin RCA to Gal-type glycans expressed by purified BJx109 and PR8. Wells coated with increasing concentrations of purified virus were probed with biotin-labeled RCA in the presence or absence of 5 mg/ml ASF. MAb 165 was used to confirm equivalent coating levels of each of the purified viruses (data not shown). (B) Binding of recombinant MGL1 to purified IAV. Wells coated with 10 μg/ml or 1.25 μg/ml of BJx109 or PR8 were incubated with 2 μg/ml recombinant MGL in buffer containing 25 mM CaCl₂ (white bars) or 5 mM EDTA (black bars). Data shown are the means from triplicate samples (± 1 SD) and are representative of 3 independent experiments. P < 0.001 (***) compared to binding to an equivalent coating concentration of BJx109 in the presence of CaCl₂. OD, optical density. (C) Monolayers of Lec1-ctrl and Lec1-MGL1 cells were mock treated or treated with 100 mU/ml of bacterial sialidase (sial) for 1 h at 37°C before infection with 10⁷ PFU of BJx109 or PR8 (i) or 7:1 viruses (ii) generated by reverse genetics. Cells were fixed at 8 h and stained for newly synthesized NP as described in the text. Data represent the mean percent infection (± 1 SD) and are expressed relative to the positive control (CHO-ctrl cells) for each virus infection. Experiments were performed 3 times with similar results. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) compared to infection of mock-treated cells by BJx109 (i) or RG-BJx109 (ii). §, percent infection was <1% of total cells.
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   FIG 7

   Mutant of MGL1 lacking 15 amino acids from its cytoplasmic domain retains C-type lectin activity but shows reduced endocytic activity when expressed by Lec1 cells. (A) Nucleotide and deduced amino acid sequences of the cytoplasmic domain of wild-type MGL1 and a deletion mutant lacking 15 amino acids from the cytoplasmic domain (ΔMGL1). The putative internalization domain YENL is underlined. (B) Cell surface expression of MGL1 on Lec1-MGL1 (black histogram), Lec1-ΔMGL1 (gray histogram), or Lec1-ctrl cells (dashed line). (C) Western blot detecting MGL in lysates from Lec1-ΔMGL1 cells. (D) MGL1 on Lec1-ΔMGL1 cells retains lectin function equivalent to that of MGL1 on Lec1-MGL1 cells. Data are representative of 2 independent experiments. (E) Reduced endocytic capacity of MGL1 expressed on Lec1-ΔMGL1 cells. MGL1-mediated endocytosis was determined as described in Materials and Methods. The asterisk indicates cell surface MAb levels that were significantly different between samples after 1 and 30 min (P ≤ 0.05; Student's t test). n.s., not significant.
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   FIG 8

   Lec1 cells expressing endocytosis-defective MGL1 show reduced sensitivity to IAV infection. (B) Binding of 5 μg/ml biotin-labeled BJx109 to cells in the presence of 10 mM CaCl₂ was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone (un). MFI (± 1 SD) of BJx109 binding to Lec1-ctrl, Lec1-MGL1, and Lec1-ΔMGL1 cells (14.4 ± 2.8, 48.4 ± 7.1, and 65.9 ± 22.5, respectively). **, P < 0.01 by one-way ANOVA compared to Lec1-ctrl. (C) Monolayers of Lec1-ctrl, Lec1-MGL1, and Lec1-ΔMGL1 were infected with 10⁷ PFU of BJx109 as described in Materials and Methods. Cells were fixed at 8 h, stained, and then examined by immunofluorescence. Data show the mean percent infection (± 1 SD) from 6 independent experiments. (**, P ≤ 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA). (D) Monolayers of CHO-ctrl (i), Lec1-MGL1 (ii), and Lec1-ΔMGL1 (iii) were incubated with 10⁷ PFU of BJx109 at 4°C for 30 min to allow virus binding and then moved to 37°C. At various times, supernatants were removed and replaced with media containing 5 mM NH₄Cl to prevent further infection. NH₄Cl was not added to mock-treated cells. All cells were fixed at 8 h and stained for expression of viral NP. Data represent the mean percent infection (± 1 SD) and are representative of 3 independent experiments. §, percent infection was <1% of total cells.

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