Journal: International Journal of Molecular Sciences

Article Title: Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

doi: 10.3390/ijms22020743

Figure Lengend Snippet: Evaluation of DC-SIGN mediated trans infection among H5N1-PVs carrying N-glycosylation mutations. ( A ) The scheme of modified conventional capture assay is demonstrated. ( B ) Raji and Raji-DC-SIGN were used as captured cells. They were incubated with H5N1-PVs at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three to five times) and the target MDCK cells were subjected to luminescence analysis. In addition, for detecting the virions budding from cis infection, the transwell system was used to monitor those virions released from captured cells further causing MDCK (target cells) infection. The lower channel of infected MDCK cells in the transwell were also subjected to luminescence analysis. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. ( C ) The Raji and Raji-DC-SIGN cells (captured cells) were incubated with H5N1-PVs carrying different N-glycosylation mutations on HA at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three-five times) and the target MDCK cells were subjected to luminescence analysis. Similarly, the detection of the virions released from cis infection of the captured cells was monitored using transwell system mentioned above. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. The significant difference was measured by each N-glycosylation mutant compared to WT group. Representative results are shown. Quantitative data represent the means ± SD of results from at least three independent experiments (WT, wild-type) (* p < 0.05; ** p < 0.01).

Article Snippet: For a DC-SIGN-enhanced infectivity assay, a 5 × 10 5 susceptible cells mentioned above were seeded into 48-well plates prior to incubation with H5N1 pseudotyped or H5N1-RG virus particles at 37 °C for 2 h. Alternatively, some of these cells were pretreated with anti-DC-SIGN monoclonal antibodies (10 μg/mL-1; R&D System, catalog no. MAB161).

Techniques: Infection, Glycoproteomics, Modification, Incubation, Cell Culture, Control, Bioprocessing, Mutagenesis