Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry ▿
- Jacqueline P. Upham1‡,
- Danielle Pickett1,
- Tatsuro Irimura2,
- E. Margot Anders1† and
- Patrick C. Reading1,3,*†
- 1Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia
- 2Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
- 3WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia
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FIG. 1.Use of VOPBA to determine direct interaction between influenza virus and the MMR. (A) Whole-cell lysate from the murine Mφ-like cell line J774E was electrophoresed by SDS-7% PAGE under nonreducing conditions, transferred to PVDF membrane, and probed with influenza virus strain HKx31. Virus was omitted from the diluent as a control for the specificity of virus binding. (B) Lysates from J774E and RAW 264.7 (RAW) cells were analyzed by VOPBA with HKx31 virus as described above. A duplicate blot was stained with polyclonal antibody raised against the MMR. (C) MMR was immunoprecipitated from J774E lysate (MMR i.p.) and resolved by SDS-7% PAGE alongside total J774E lysate precleared with protein G-Sepharose (J774E), and the lysate postimmunoprecipitation (post i.p.). A VOPBA with virus strain HKx31 was performed. The approximate molecular mass positions are indicated in kilodaltons.
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FIG. 2.Mode of interaction of influenza virus with the MMR. J774E Mφ proteins were sialidase-treated (+) or not treated (−) prior to electrophoresis on SDS-7% PAGE and electrotransfer. Blots were probed with influenza virus in a VOPBA (A and B) or immunoblotted with anti-MMR IgG (C). The viruses used in the VOPBA were HKx31 (A) and Guangdong/93 (B), and the buffer used was BB-Ca 2+ buffer as described in Materials and Methods, except for the lanes marked “EDTA,” in which the virus-binding step was carried out in BB buffer lacking CaCl2 and supplemented with 5 mM EDTA. The approximate molecular mass positions are indicated in kilodaltons.
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FIG. 3.Influenza virus binds to MGL. (A) Comparison of Mφ proteins recognized by influenza virus and by anti-MGL antibody. J774E Mφ lysate that had been sialidase-treated (+) or not treated (−) was resolved in duplicate by SDS-10% PAGE, and blots were probed with virus (Guangdong/93) in a VOPBA or immunoblotted with MAb LOM-8.7 to detect MGL. (B) MGL was isolated from J774E Mφ by affinity chromatography on ASF (MGL) and resolved by SDS-10% PAGE alongside J774E cell lysate (Mφ). The binding of Guangdong/93 virus was examined in VOPBA using virus binding buffer NiBB-Ca2+ (control) or the same buffer supplemented with 0.1 M galactose or 0.1 M mannose. The approximate molecular mass positions are indicated in kilodaltons.
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FIG. 4.Coprecipitation of Mφ cell surface proteins with influenza virus after ultracentrifugation. J774E cells were surface labeled with biotin prior to cell lysis. Influenza virus (Guangdong/93) was incubated with the lysate in the presence of 10 mM Ca2+ or 5 mM EDTA and then precipitated by ultracentrifugation and washed. Final pellets were boiled and then resolved by SDS-12% PAGE and blotted with streptavidin. Samples of biotinylated lysate only (Mφ) and virus only (virus) were subjected to the same treatment and served as controls. The approximate molecular mass is indicated in kilodaltons.
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FIG. 5.Expression of MMR and MGL by Mφ correlated with ability of mannan and/or ASF to inhibit infection by influenza virus. (A) Expression of MMR and MGL on J774E Mφ, RAW 264.7 Mφ (RAW) or LA-4 epithelial cells. Cells were stained with biotin-labeled MAbs to MMR or MGL (shaded histograms) or with appropriate biotin-labeled isotype control MAbs (open histograms). A minimum of 20,000 cells were collected for analysis. (B) Monolayers of J774E Mφ, RAW 264.7 Mφ or LA-4 epithelial cells were incubated for 30 min at 37°C in serum-free medium alone or supplemented with increasing concentrations of either mannan or ASF, as indicated, prior to the addition of 106 PFU of Guangdong/93. Mannan or ASF were included during subsequent culture of the cells. Cells were fixed 6 to 8 h postinfection and stained for the expression of influenza virus NP, and the percentage of virus-infected cells was determined via immunofluorescence. The data represent the mean percent infection ± 1 standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01 (percent infected cells in the presence of inhibitor that were significantly different from the percent infected cells recorded in the absence of either inhibitor as determined by one-way ANOVA).
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FIG. 6.The poor ability of strain PR8 to infect Mφ correlates with weak interactions with MMR and MGL. (A) Susceptibility of Mφ and epithelial cell lines to infection by Guangdong/93 (□), HKx31 (▦), and PR8 (▪) influenza viruses. Alv, resident alveolar Mφ. Monolayers of cells were infected in chamber slides with 106 PFU of influenza virus as described in Materials and Methods. The percentage of infected cells was determined in five independent fields per chamber by fluorescence microscopy. The data are expressed as mean percent infected cells ± 1 standard deviation and are representative of at least two independent experiments. *, P < 0.01 (exposure of cell monolayers to PR8 resulted in a significantly lower percentage of infected cells compared to cell monolayers exposed to either Gundaong/93 or HKx31 [one-way ANOVA]). (B) Comparison of Mφ proteins bound by PR8 and Guangdong/93 strains of influenza virus in VOPBA. (i) Samples of J774E lysate (Mφ) that had been sialidase treated (+) or not treated (−) were resolved by SDS-7% PAGE prior to blotting and application of virus. (ii) J774E lysate and isolated MGL, obtained by ASF-Sepharose affinity chromatography of lysate, were resolved by SDS-10% PAGE prior to blotting and application of virus.
- American Society for Microbiology
