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.