Fig. 2: HLA-DR and SLA-DR allow FLUAV entry into mammalian cells.

a HEK-293T cells were transfected with either HLA-DRA + HLA-DRB1 or SLA-DRA + SLA-DRB1. MHCII-expressing cells and cells transfected with an empty plasmid were desialylated at 24 h post-transfection for 24 h. Cells were infected with either sOH/04 or hVIC/11 and infections were incubated for 24 h before processing. Subsequently, cells were stained for MHCII (pink), sialic acid (red), and FLUAV (green). Co-localization between MHCII and FLUAV can be seen as white. Scale bar= 30 μm. Images are representative of three independent experiments. Transient expression of MHCII allows FLUAV entry into desialylated HEK-293T cells. Cells were transfected with either HLA-DR (b) or SLA-DR (c) and further desialylated prior to infection with hVIC/11 or sOH/04. At 24 hpi, cells were fixed and stained for MHCII and FLUAV. The percentage of FLUAV-positive cells was determined by flow cytometry (n = 3 independent experiments). d Percentage of FLUAV-positive cells among desialylated MHCII-expressing cells determined by flow cytometry represented as the mean ± SEM of three independent experiments. sOH/04 is shown in blue while hVIC/11 can be seen in orange. e Percentage of infected HEK-293T cells (regardless of MHCII expression) determined by flow cytometry. Data are presented as the mean ± SEM of three independent experiments. f MHCII-expressing cells were incubated with 20 mM NH₄Cl for 1 h prior to infection with either sOH/04 (blue) or hVIC/11 (orange) at an MOI of 1. Infections were maintained at 37 °C for 24 h in presence of NH₄Cl and intracellular vRNA was determined at 24 hpi by RT-qPCR. Data are presented as the mean ± SEM of n = 3 independent experiments. g Transfected HEK-293T cells were incubated with 10 nM Bafilomycin A1 before infection and were subsequently infected at an MOI of 1 with sOH/04 (blue) or hVIC/11 (orange). Intracellular FLUAV vRNA was quantified at 24 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D. limit of detection. Untreated: non-transfected, non-desialylated cells. Empty vector: desialylated cells transfected with an empty pCAGGS plasmid. HLA-DR: desialylated cells expressing HLA-DR. SLA-DR: desialylated cells expressing SLA-DR.