Fig 2: ALPP directly interacts with ZIKV proteins and prevents their proteasomal degradation. (A and B) Immunoblot analyses of the ZIKV protein levels. WT and ALPP−/− JEG-3 cells were transiently transfected with pCMV-ZIKV nonstructural proteins for 24 h and treated without (A) or with (B) MG132 (10 μM) for an additional 12 h. (C) Immunoblot (left) and immunoprecipitation (right) analyses of lysates from WT and ALPP-overexpressing U-251 MG cells infected with ZIKV for 24 h. (D) Immunofluorescence analyses of the colocalization between ALPP and ZIKV proteins. Flag-ALPP U-251 MG cells were infected with ZIKV for 24 h (MOI = 2). Scale bars, 5 μm (middle row) and 10 μm (top and bottom rows). ALPP, red; ZIKV C, ENV, or NS2B, green.

Fig 3: ALPP functions at multiple steps in the ZIKV infectious life cycle. (A) Schematic of ZIKV infection experiments comparing WT and ALPP−/− JEG-3 cells. Capital letters in parentheses correspond to panels B to E of the figure. (B) Detection of ZIKV vRNA by RT-qPCR. Statistical analyses of internalized ZIKV were performed; MOI = 100. The data were obtained from four biological replicates (n = 4). n.s., no significance (P = 0.1934; two-tailed Student's t test). (C) Detection of intracellular ZIKV vRNA at the indicated times after ZIKV infection by RT-qPCR. MOI = 0.5. The data were obtained from four biological replicates (n = 4). The significant differences between WT and ALPP−/− JEG-3 cells at each time point were determined by two-way ANOVA with Sidak’s multiple-comparison test. ****, P < 0.0001; n.s., no significance; ND, not detected. (D) ZIKV MR766 (left) and SZ01 (right) infectious particles produced from WT and ALPP−/− JEG-3 cells at the indicated time after ZIKV infection. MOI = 0.5. The data were obtained from at least three biological replicates (n ≥ 3). Significant differences between WT and ALPP−/− cells at each time point were determined by two-way ANOVA with Sidak’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. TCID50, 50% tissue culture infective doses. (E) Production of ZIKV MR766 (top) and SZ01 (bottom) proteins in WT and ALPP−/− JEG-3 cells infected with ZIKV for 24 h. MOI = 0.5. The quantitative data in this figure are shown as means ± SEMs.

Fig 4: ALPP depletion prevents productive ZIKV infection in human placental trophoblast cells and astrocytes. (A and B) ZIKV infection in WT and ALPP-KO U-251 MG astrocytoma cell lines. (A) Representative immunofluorescence images from three independent experiments. MOI = 0.5. Scale bars, 200 μm. (B) Statistical analyses. Each biological replicate (n = 3) contained 3,000 analyzed cells. ****, P < 0.0001 (significantly different compared with the WT cells; two-way ANOVA with Sidak’s multiple-comparison test). (C and D) Immunoblot (C) and immunofluorescence (D) analyses of ALPP protein levels. The images shown are representative of results from three independent experiments. Scale bars, 200 μm. ALPP, green; DAPI, blue. (E and F) ZIKV, IAV, and DENV infection in WT and ALPP−/− human placental trophoblasts (JEG-3). (E) Representative immunofluorescence images from three independent experiments. Scale bars, 100 μm. (F) Statistical analyses. Each biological replicate (n = 3) contained 3,000 analyzed cells. n.s., no significance; **, P = 0.0015; ****, P < 0.0001 (significantly different compared with the WT cells; two-tailed Student's t test). (G) ZIKV infection in ALPP-overexpressing ALPP−/− JEG-3 cells and 293T cells. MOI = 2. Each biological replicate (n = 3) contained 3,000 analyzed cells. ****, P < 0.0001 (significantly different compared with the NC-treated cells; two-tailed Student's t test); **, P = 0.0052 (significantly different compared with the NC-treated 293T cells; two-tailed Student's t test). The quantitative data in this figure are shown as means ± SEMs. DAPI, blue; ZIKV E, red; NP, red; DENV, red; NC, negative control.

Fig 5: The catalytic activity and the C terminus of ALPP are needed for its function in promoting ZIKV infection. (A) Residues that have been mutagenized in ALPP activity sites. A colored box over the residue number indicates that it is a ligand to the active site Mg (blue), Zn1 (yellow), Zn2 (purple), or both Zn2 and Mg (red). (B) Statistical analyses of ZIKV infection in wild-type ALPP- or alkaline phosphatase-impaired ALPP mutant-overexpressing cells. Each biological replicate (n ≥ 3) contained 3,000 analyzed cells. MOI = 0.5. n.s., no significance; *, P < 0.05; **, P < 0.01 (significantly different compared with the wild-type ALPP-overexpressing cells; two-tailed Student's t test). (C) Structures of the wild-type, mutant, and delta-C-terminal ALPP used in this study. (D) Statistical analyses of ZIKV infection in cells overexpressing wild-type or mutant ALPP. Each biological replicate (n ≥ 3) contained 3,000 analyzed cells. MOI = 0.5. n.s., no significance; ***, P < 0.001; ****, P < 0.0001 (significantly different compared with the wild-type ALPP-treated cells; two-tailed Student's t test).