Figure 4

Flagellin augments pseudoparticle infection post-attachment. (a) To assess whether flagellin can directly bind VSV-Gpp the virus was incubated with histidine tagged flagellin (0.3 μg/mL) for 1 h, complexes captured with Ni-NTA agarose beads and viral associated RTase activity measured. (b) To determine whether flagellin augments virus attachment A549 cells were treated with flagellin (0.3 μg/mL) at 37 °C for 1 h and inoculated with VSV-Gpp for 4 °C or 37 °C for 1 h, unbound virus removed by extensive washing and cell-associated viral RTase activity measured. (c) To ascertain whether flagellin can promote infection post virus-cell attachment, A549 cells were incubated with VSV-Gpp for 1 h at 4 °C or 37 °C, unbound virus removed by washing and cells treated with flagellin (0.3 μg/mL) and cultured for 48 h before measuring infectivity. (d) Schematic diagram outlining the principles of the MeV cell-cell fusion assay. To determine fusion, MeV F and H glycoproteins are expressed in effector cells along with an enzymatically inactive split GFP-renilla luciferase. In parallel, the receptor SLAMF1 is delivered into target cells with the remaining half of the GFP-renilla reporter. After co-culturing effector and target cells, F and H receptor engagement triggers cell-cell fusion, GFP-Renilla reconstitution and reporter gene activity. (e) Target cells were treated with STm FliC (1 μg/mL) for 1 h and co-cultured with effector cells for 24 h and lysed to read luciferase activity. (f) A549-SLAM cells were treated with flagellin (1 μg/mL) for 1 h and infected with Measles virus (MOI 0.1) for 24 h, lysed and the infectivity of cell-associated virus determined (tissue culture infectious dose - TCID₅₀/mL). Bars represent mean ± S.D. for n = 3. Statistical comparison by unpaired t test where: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001. All data sets are representative of at least two independent experiments.