Involvement of the Mannose Receptor in Infection of Macrophages by Influenza Virus
- Patrick C. Reading † ,
- Joanna L. Miller, and
- E. Margot Anders *
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Fig. 1.Binding of 125I-mBSA to peritoneal Mφ. Mφ were preincubated on ice for 20 min with binding buffer alone or supplemented with additives as indicated, before the addition of 5 × 105 cpm of 125I-mBSA. Results are expressed as the mean specific binding (and 1 standard error) of125I-mBSA in triplicate samples as described in Materials and Methods.
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Fig. 2.Relationship between the relative binding avidity of the virus HANA glycoproteins for the MR on peritoneal Mφ and the content of high-mannose and hybrid-type oligosaccharide on influenza virus as determined by 125I-ConA binding. The virus strains used were BJx109 (■), HKx31 (⊕), and PR8 (▵). (A) Ability of HANA preparations of the three viruses to inhibit the binding of 125I-mBSA to peritoneal Mφ. (B) Binding of125I-ConA to microtiter wells coated with increasing concentrations of purified influenza viruses. Binding was completely inhibited in the presence of 100 mM α-methylmannoside (data not shown). Coating levels of the three viruses were confirmed to be similar by using MAbs to the viral NP and to the host-derived carbohydrate antigen common to all egg-grown strains of influenza virus, as described in Materials and Methods (data not shown).
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Fig. 3.Mannan inhibits infection of peritoneal Mφ by influenza virus. (A) Infection of peritoneal Mφ by 106PFU of influenza viruses BJx109, HKx31, and PR8 in the absence (■) or presence (□) of 5 mg of mannan per ml. Mannan was present both at the time of virus adsorption and during subsequent culture of the Mφ. (B) Effect of the time of addition of mannan on inhibition of infection of Mφ by BJx109 virus. Results are presented as the mean percent infection (and 1 standard error) from three experiments.
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Fig. 4.Comparison of J774 and J774E cells for MR expression and susceptibility to influenza virus infection. (A) Binding of125I-mBSA to J774 (◊) and J774E (⧫). Scatchard plots of the data (inset) yielded Kd values of 10 and 11.5 nM for J774 and J774E cells, respectively. For these calculations, the molecular mass of mBSA (31 mol of mannose per mol of BSA) was taken to be 73,000 kDa. (B) Infection of J774 (open symbols) and J774E (solid symbols) by BJx109 (□, ■), HKx31 (○, ●), and PR8 (▵, ▴). The greater susceptibility of J774E cells to influenza virus infection was observed in multiple experiments.
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Fig. 5.Downregulation of MR in J774E cells is associated with reduced susceptibility to influenza virus infection. (A) J774E cells were cultured for 2 h in the presence (◊) or absence (⧫) of 25 mM d-mannose, then washed free of the sugar and assayed for125I-mBSA binding. Scatchard plots of the data (inset) yielded Kd values of 10 and 11.1 nM for cells cultured in the presence or absence of mannose, respectively. (B) J774E cell monolayers in chamber slides were incubated for 2 h in the presence (□) or absence (■) of 25 mM d-mannose and then infected for 1 h with increasing doses of BJx109. The presence or absence of mannose was maintained, as appropriate, throughout all stages of the infection assay including virus adsorption, washing, and subsequent culture steps. The results given are from one of two similar experiments.
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Fig. 6.Role of sialic acid in the interaction of influenza virus glycoproteins with the MR. Peritoneal Mφ were incubated with V. cholerae type III NA (▨) or in medium alone (mock treated) (■) for 60 min at 37°C, washed, and used in binding assays with 125I-BJx109 HANA (A) and125I-mBSA (B). Specific binding of 125I-mBSA was determined in the absence or presence of 10 μg of unlabeled BJx109 HANA per ml. Results are expressed as mean cpm bound (and 1 standard error) from triplicate samples and are from one of two similar experiments.
- Copyright © 2000 American Society for Microbiology
