Fig 1: Galectin-1 on the cell surface mediates ZIKV attachment and infection of host cells(A) Cell surface expressed BioID2-RBP(Z)-GPI chimeric protein in human neural progenitor cells (HNPCs) was PI-PLC sensitive. HNPCs transfected with BioID2-RBP(Z)-GPI was stained with an antibody specific for HA or control IgG at the same concentration showing that the chimeric protein was localized on the cell surface. Nuclei were counterstained with DAPI. Flow cytometry analysis was performed for cells treated with or without PI-PLC and stained with an antibody specific for HA. BG: background (in shadow), HNPCs transfected with the empty vector. Scale bar: 40 µm.(B) Cell surface expressed BioID2-RBP(Z)-GPI chimeric protein in HNPCs was able to biotinylate proteins in the presence of exogenously added biotin. Immunoblotting with streptavidin-HRP showed that in the presence of exogenously added biotin, many more proteins were labeled in HNPCs expressing BioID2-RBP(Z)-GPI chimeric protein than in HNPCs transfected with the empty vector. Silver staining of the SA-beads purified proteins in the presence or absence of biotin showed that the differences were consistent. Some of the differentiated purified protein bands (indicated by the red arrow) were excised and subjected to mass spectrometry analysis.(C) Galectin-1 could be detected on the cell surface of A549 and MNCs. Immunofluorescence staining with an antibody specific for galectin-1 (red) showed that galectin-1 could be detected on the cell surface although a significant amount of galectin-1 could also be detected in the cytosol of A549 cells. Scale bar: 40 µm.(D) The binding between galectin-1 and E protein (envelop protein of ZIKV) is carbohydrate-dependent. Schematic diagrams of FLAG-tagged ZIKV E protein (ZIKV-E-FLAG), HA-tagged ZIKV E protein (ZIKV-E-HA), HA-tagged N154Q ZIKV E protein (ZIKV-E(N154Q)-HA), FLAG-tagged human galectin-1, and HA tagged mouse galectin-1. Numbers represent the number of amino acids of each component. These chimeric proteins were expressed in HEK293 T cells as indicated. Co-immunoprecipitation was performed and interactions between galectin-1 and E protein were detected with an antibody specific against HA or FLAG. The light chain of the antibody used for co-immunoprecipitation was indicated.(E) Attachment assay for ZIKV on A549 cells showed that Zika virions were encapsulated by galectin-1 on the cell surface (indicated by the arrow). ZIKV used at 100 MOI was incubated with A549 cells at 4°C for 5 min. Confocal immunofluorescence staining of ZIKV E protein (green) or galectin-1 (red) was performed using antibodies specific for ZIKV E protein or galectin-1, respectively. Phase contrast image was also taken at the same time to show the edge of cells. Scale bar: 40 µm or 10 µm as indicated.(F) Silencing galectin-1 reduces ZIKV attachment to A549 cells. mRNA for ZIKV was extracted from A549 cell expressing galectin-1 or not expressing galectin-1 and RT-qPCR was performed to quantify the mRNA level of ZIKV E protein. Virions were incubated with the A549 cells at 4°C for 1 h. NC: knockout control; 2#&3# were two different targets for the human galectin-1 knockout. Data are represented as mean +/- SEM.(G) Down-regulation of galectin-1 in MNCs significantly decreased ZIKV attachment to MNCs. ZIKV was used at MOI = 100 and incubated with MNCs at 4°C for 1 h. Total mRNA was extracted and ZIKV E protein mRNA was quantified by RT-qPCR. NC: empty vector shRNAi. 1#, 2#, and 3# were different target sites for mouse Lgals1. Data are represented as mean +/- SEM.(H) Downregulation of galectin-1 in MNCs significantly decreased ZIKV E protein levels. Galectin-1 expressing and not expressing MNCs were incubated with ZIKV at MOI = 0.01 and cultured for 72 h. Galectin-1 and ZIKV E protein levels were detected by specific antibodies. Actin was blotted as a loading control. IMAGE J was used to quantify relatively ZIKV E protein to actin. NC, 1#, 2#, and 3# were described as in Figure 4G. Data are represented as mean +/- SEM.(I) Antibodies specifically against galectin-1 blocked ZIKV infection of A549 cells. cells were pretreated for 30 min with mouse monoclonal antibodies (Orb627212 and MBS769547) against galectin-1 or isotype control IgG1 used at the same concentration. The cells were then challenged with ZIKV. Blocking efficacy was calculated as follows: (mean pixel from control antibody treated cells minus mean pixel galectin-1 antibody treated cells)/mean pixel from control antibody-treated cells x 100%. Pixel was determined using IMAGE J. Data are represented as mean +/- SEM.(J) Down-regulation of galectin-1 in MNCs significantly decreased ZIKV infection of MNCs but did not impact virus releasing from host cells. Relative extracellular virus titer and intracellular virus titers were quantified by RT-qPCR based upon ZIKV E protein mRNA (ZIKV E protein mRNA at 5 × 103 copy was arbitrarily defined as 1). Infection of galectin-1 expressing and not expressing cells was performed at MOI = 0.01 for 72 h. Generation of NC, 1#, 2#, and 3# were described as in 4G. Data are represented as mean +/- SEM.(K) Down-regulation of galectin-1 in MNCs significantly mitigated mature Zika virion formation based on plaques assays. Zika virions collected from the culture medium of MNCs expressing or not expressing galectin-1 were serially diluted for plaque assay. 1 × 104 virion/mL was defined as 1 PFU. The bar graph showed the number of plaques formed. p-value and number of independent experiments were indicated. NC, 1#, and 3# were described as in Figure 4G. Data are represented as mean +/- SEM. See also Figure S4.