Fig 2: The detection of ITGB4 associates with ZIKV E.a Hun-7 cell lysates were incubated with ZIKV-E or RuV-E1 proteins and the affinity-isolated proteins were subjected to SDS-PAGE. The red arrowhead indicates the specifically isolated protein using the ZIKV-E protein (n = 3 of independent experiments). b ITGB4-derived tryptic fragments as determined by mass spectrometry. The tryptic fragments corresponding to ITGB4 protein sequence (Gene bank: NM_000213) is indicated in red. c Immunoprecipitation assay showing specific binding between ZIKV-E and the ectodomain of ITGB4. Lysates of 293 cells were incubated with ZIKV-E or RuV-E1 immobilized on anti-Flag M2 monoclonal antibody-conjugated agarose beads. The precipitates were washed by DDM-PBS and eluted with Flag peptide. The eluents were then separated by SDS-PAGE and immunoblotted with the related antibodies (n = 3 of independent experiments). d Immunoprecipitation assay showing specific binding between ZIKV-E and ITGB4/ITGA6 heterodimer in Hun-7 cells. Lysates of Hun-7 cells were incubated with ZIKV-E or RuV-E1 immobilized on anti-FLAG M2 monoclonal antibody-conjugated agarose beads. The precipitates were treated as in (c) (n = 3 of independent experiments). CL: Hun-7 cell Lysates. e ZIKV-E protein interacted with ITGB4. Purified ITGB4, ITGB4/ITGA6 or BSA were coated on 96-well plate overnight and incubated with flag tagged ZIKV-E or RuV-E1 for 3 h at 4°C. ZIKV-E or RuV-E1 was detected by HRP-conjugated anti-Flag antibody. The data were expressed as Mean ± SD of three independent experiments. **p = 0.0057, ****p < 0.0001, two-sided, Student’s t test. f ZIKV virions interacted with the ectodomain of ITGB4. Purified ITGB4, ITGB4/ITGA6 or BSA was coated on 96-well plate overnight and incubated with MR766 or Mock (PBS) for 4 h at 4°C. The bound virus was detected by HRP-conjugated anti-E antibody. The data were expressed as Mean ± SD of three independent experiments. ***p = 0.0002, **p = 0.032, two-sided, Student’s t test. g, h Susceptibility of Vero, Hun-7, A549, LLC-MK2, hNPCs and HEK-293T cells to ZIKV. Cells were infected with ZIKV MR766 at an MOI = 0.05. 48 h post infection and then visualized using a human antibody that recognizes the structural protein E (green). Cell nuclear were stained with DAPI (blue). n = 3 of independent experiments. All scale bars are 50 μm.

Fig 3: Generation and characterization of iPSC-derived EpSCs. a Schematic of the EpSC differentiation showing the presence of retinoic acid, BMP-4, and EGF at different times. b Morphology of cells at different stages of differentiation: iPSCs, high-density polygonal morphology of cells differentiated for 11 days, and paving stone morphology of cells differentiated for 18 days. Scale bar, 50 μm. c Percentage of positive cells were 20.73 ± 7.26%, 20.05 ± 6.02%, 18.57 ± 4.59%, 17.71 ± 6.19%, 17.90 ± 4.42%, 22.94 ± 6.37%, and 0.01% ± 0.01% for ITGA6, CD200, Krt14, Krt15, ITGB1, Krt19, and NANOG respectively (n = 5, mean ± SD). d Immunofluorescence analysis showing positive expression of CD200, Krt15, Krt19, ITGB1, and ITGA6 and negative expression of NANOG in differentiated cells in comparison with iPSCs. Scale bar, 30 μm. e–g RT-PCR analyses of epithelial stem cell-related genes (LGR5, LGR6, TCF4, FZD2, DDK3, CTNNB1, Krt14, LEF1, and LHX2) (e), hair follicle stem cell-related genes (CD200, Krt15, Krt19, and ITGA6) (f), and pluripotent genes (NANOG, OCT4, and REOX1) (g) in cells induced for 11 days compared with control hair follicle stem cells (hHFSCs) and iPSCs. The housekeeping gene GAPDH was used as an internal reference. Error bars represent the S.D. (n = 3). h Flow cytometric analysis of Krt14 in iPSC-derived EpSCs in comparison with iPSCs. i Flow cytometric analysis of CD200 and ITGA6 in iPSC-derived EpSCs in comparison with iPSCs

Fig 4: Soluble ITGB4 and ITGB4/ITGA6 inhibit ZIKV MR766 infection.a Western blot analysis of the ZIKV in 293 T cells transfected with ZIKV genome RNA. 2 ug purified ZIKV genome RNA was transfected into 293 T cells for 24 h. Then, the supernatant of 293 T cells was transferred into Vero cells (n = 3 of independent experiments). Control: Vero cells infected with ZIKV for 24 h. Mock: Vero-E6 cells. b Viral titers in cell supernatants were determined by TCID50 experiment in Vero-E6 cells. The data were expressed as Mean ± SD of five independent experiments. *P = 0.0204, two-sided, by multiple comparisons by One-way ANOVA test. Control: Vero cells infected with ZIKV for 24 h. 293T-ZIKV: Vero cells incubated with the supernatant of 293 T cells transfected with ZIKV genome RNA. 293 T: Vero cells were incubated with the supernatant of 293 T cells. c Cells were subsequently inoculated for 1 h with the ZIKV MR766-protein mixes on ice followed washing three times with PBS. The virus titer was determined by plaque assay on Vero cells at 48 h after infection. The data were expressed as Mean ± SD of three independent experiments. ****p < 0.0001 (Vero), ****p < 0.0001 (A549), **P = 0.0045 (Hun7), ***P = 0.0002 (Hun7), ****p < 0.0001 (MK2), **P = 0.0013 (hNPCs), ****p < 0.0001 (hNPCs), two-sided, multiple comparisons by One-way ANOVA test. d Cells were infected with ZIKV as described above. After incubated with virus-protein mixes for 1 h on ice, cells were washed with cold PBS. Total RNA was then extracted and ZIKV RNA levels that represent viral particles bind to cell surface were quantified by RT-PCR assay. The GAPDH was used as internal control gene. The data were expressed as Mean ± SD of three independent experiments. Control: PBS buffer. **P = 0.0038 (Hun7, top), ***P < 0.0003 (Hun7, middle), *P = 0.0241 (Hun7, bottom), **P = 0.0022 (Vero, top), *P = 0.0475 (Vero, bottom), *P = 0.0117 (MK2), **P = 0.0072 (A549, top), *P = 0.0006 (A549, middle), *P = 0.0072 (A549, bottom), *P = 0.0102 (hNPCs, top), ***P = 0.0002 (hNPCs, middle), **P = 0.0032 (hNPCs, bottom), two-sided, multiple comparisons by One-way ANOVA test. e ZIKV MR766 was preincubated with the indicated concentrations of soluble ITGB4/ITGA6 or BSA. Cells were subsequently inoculated for 1 h with the virus-protein mixes on ice. Cells were washed three times with PBS and the virus titers were determined by plaque assay on Vero cells at 48 h after infection. The data were expressed as Mean ± SD of three independent experiments. ***P = 0.003 (Vero), ****p < 0.0001 (Vero), ***P = 0.0001 (A549), ****p < 0.0001 (A549), **P = 0.0022 (Hun7), ***P = 0.0002 (Hun7), **P = 0.0034 (MK2), ***p = 0.0007 (MK2), *P = 0.0257 (hNPCs), **P = 0.0016 (hNPCs), two-sided, multiple comparisons by One-way ANOVA test. f Cells were infected with ZIKV that preincubated with the indicated concentrations of soluble ITGB4/ITGA6 or BSA. Incubated with virus-protein mixes on ice for 1 h, cells were washed three times with cold PBS. Total RNA was then extracted and ZIKV RNA levels were determined by RT-PCR assay. The data were expressed as Mean ± SD of three independent experiments. Control: PBS buffer. **P = 0.0055 (Hun7, top), ***P = 0.0002 (Hun7, middle), **P = 0.0099 (Hun7, bottom), *P = 0.0197 (Vero, top), **P = 0.0017 (Vero, bottom), *P = 0.0227 (MK2, top), **P = 0.0011 (MK2, middle), *P = 0.0403 (MK2, bottom), **P = 0.0061 (A549, top), ***P = 0.0001 (A549, middle), **P = 0.0033 (A549, bottom), **P = 0.0064 (hNPCs, top), ***P = 0.0004 (hNPCs, middle), *P = 0.0232 (hNPCs, bottom), two-sided, multiple comparisons by One-way ANOVA test.

Fig 5: Inhibition of ZIKV infection by antibody to ITGB4.a A surface plasmon resonance assay (SPR) characterizing the binding between ITGB4/ITGA6 and 13H10 antibody. The 13H10 antibody were immobilized on the chip at about 300 response units. Concentrations of ITGB4/ITGA6 was used to flow over the chip surface. 13H10 was ITGB4 specific antibody. b Cells were treated with various concentrations of 13H10 or isotype antibody for 1 h and infected with ZIKV with MOI = 0.05. Virus titers were determined by plaque assay on Vero cells at 48 h after infection. The values in the graph represent the mean ± SD of n = 3 independent experiments. ***P = 0.0082 (Vero), ****p < 0.0001 (Vero), **P = 0.0052 (A549), ***p = 0.0005 (A549), ***P = 0.0002 (Hun7), ****p < 0.0001 (Hun7), ****p < 0.0001 (MK2), **P = 0.0046 (hNPCs), ***P = 0.0002 (hNPCs), two-sided, multiple comparisons by One-way ANOVA test. c Cells were preincubated with isotype antibody or indicated concentrations of 13H10 antibody for 1 h and then challenged with ZIKV with MOI = 10 at 4 °C for 1 h. Cells were washed, and total RNA was extracted. ZIKV RNA was quantified by RT-PCR. The results were expressed as ZIKV RNA levels relative to the expression of the GAPDH internal control gene. The data were expressed as Mean ± SD of three independent experiments. ****P < 0.0001 (Hun7, top), ***P = 0.0001 (Hun7, middle), **P = 0.0042 (Hun7, bottom), ***P = 0.0002 (Vero, top), **P = 0.0019 (Vero, middle), *P = 0.0192 (Vero, bottom), ****P < 0.0001 (A549, top), ****P < 0.0001 (A549, middle), **P = 0.0029 (A549, bottom), ***P = 0.0002 (MK2, top), **P = 0.0025 (MK2, middle), *P = 0.0216 (MK2, bottom), ***P = 0.0003 (hNPCs, top), **P = 0.0060 (hNPCs, middle), *P = 0.0169 (hNPCs, bottom), two-sided, multiple comparisons by One-way ANOVA test.