Fig. 3: GII.4 infection in HIEs is sensitive to effectors of CLIC pathway.

a Schematic of macropinocytosis and the Clathrin Independent Carrier (CLIC) pathways, their effectors and inhibitors. b GII.4 replication in the presence of EIPA (Na⁺/H⁺ exchanger inhibitor), ML141 (Cdc42 inhibitor), Wiskostatin (N-WASP inhibitor), NSC23766 (RAC1 inhibitor), CT04 (RhoI inhibitor), Blebbistatin (myosin inhibitor), and LY29402 (PI3K inhibitor) at 1 h (gray) and 24 h (orange). c GII.4 replication in the presence of Arf1 inhibitor Golgicide A (GCA at 1 h (black) and 24 h (blue). d GII.4-induced tubular carriers at 1 h (37 ^oC) detected using guinea pig anti-Sydney VLP polyclonal antibody (Gp Syd-pAb) for viral capsid (green) and phalloidin for actin (red). Images were taken using a ZEISS Laser Scanning Microscope LSM 980 with Airyscan 2. e Electron microscopy to identify CLIC structures in GII.4 VLP-treated HIEs. (1–7 structures/cell) compared to, mock-treated cells (no structures in n = 25 cells). f Confocal microscopy to detect GII.4 VP1 capsid (green) colocalization with gal-3 (red) on the cell surface at 10 min and 1 h (37 ^oC) after VLP treatment using anti-gal-3 and Gp Syd-pAb (n = 3 HIE replicates). g Effect of blocking GII.4 virus-galectin-3 (gal-3) surface interaction using anti-gal-3 antibody on GII.4 replication at 1 h (black) and 24 h (red). h Dot blot analysis investigating GII.4 VLP interaction with purified gal-3. i Probing CLIC carriers utilized in HIEs for endocytosis with Alexa Fluor™ 594 conjugated cholera toxin B (CTxB) (red) and GII.4 VLPs (green) with similar cargoes marked by white arrows (n = 2 HIE replicates). Inset: Co-occurrence of CTxB and GII.4 VLPs in similar cargos, scale = 5 µm. All the experiments were repeated independently three times with similar results. In b, c, and g viral GEs were quantified using n = 2 independent HIE replicates for the 1 h and n = 3 independent HIE replicates for 24 h with two technical replicates/sample. The error bars represent mean ± SD with P values calculated using one-way ANOVA, Dunnett’s multiple comparisons test with comparisons at 24 h relative to untreated control. Source data are provided as a Source Data file.