Journal: Cellular and Molecular Life Sciences
Article Title: Glucosylceramide in bunyavirus particles is essential for virus binding to host cells
doi: 10.1007/s00018-023-05103-0
Figure Lengend Snippet: Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL- threo -phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A , and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP ( n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR ( n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor DC-SIGN (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied ( n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using phycoerythrin-conjugated anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer ( n = 4)
Article Snippet: The location of DC-SIGN was assessed at the surface of BHK-21 cells (not permeabilized) by flow cytometry using an anti-DC-SIGN phycoerythrin-conjugated antibody (FAB1621P R&D Systems) according to a standard procedure [ ].
Techniques: Virus, Binding Assay, Derivative Assay, Western Blot, Produced, Quantitative RT-PCR, Labeling, Infection, Flow Cytometry, Expressing, Control, Comparison, Fluorescence, Transduction, Retroviral, Plasmid Preparation, Incubation, Immunostaining
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![Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL- threo -phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A , and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP ( n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR ( n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor <t>DC-SIGN</t> (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied ( n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using <t>phycoerythrin-conjugated</t> anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer ( n = 4)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4583/pmc10834583/pmc10834583__18_2023_5103_Fig7_HTML.jpg)