Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

Abstract

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV⁺ pericytes of fetal human brain specimens and in vertically transmitted ZIKV⁺ human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.

Extended Data Fig. 1. Perinatal ZIKV infection in brain tissues of deceased fetuses exhibits pronounced calcifications.

(a) Paraffin-embedded brain tissue sections (10μm) from ZIKV-positive deceased human fetuses were stained with Von Kossa (red arrow). (b) Immunohistochemical staining for NeuN (neurons), PDGFRβ (pericytes) or GFAP (astrocytes) were performed using sequential brain sections of ZIKV-positive human fetal brain tissue. Arrows indicate NeuN-, PDGFRβ- or GFAP-positive cells (c) RNAScope duplex in-situ hybridization with ZIKV RNA and PDGFRβ mRNA of healthy human fetal brain tissues. Black arrows indicate PDGFRβ-positive cells lining the blood vessels. Neither non-specific staining ZIKV RNA nor calcium deposit was detected in healthy tissues. Data in a-c are examined from biologically independent human specimens (healthy n=3; ZIKV n=5) and are representative of two independent repeats.

Extended Data Fig. 2. Asian ZIKV infection, but not African ZIKV elicits in vitro calcification in pericytes.

Human fetal pericytes were infected with different ZIKV strains at MOI of 0.5 or mock (PBS) as control (n=4 biologically independent cells per group). (a) At 3 dpi, cell viability was quantified. See Supplementary Data Fig. 1 for gating strategy. (b) BMP4, BMP6, BMP7, BMP9 and NOG expression between ZIKV MR766- and ZIKV H/PF/2013-infected pericytes at 1, 3 and 4 dpi (n=6 biologically independent cells per group). Data in a and b are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. (c) Osteogenic gene expression between ZIKV MR766- and ZIKV IbH30656-infected pericytes was normalized to GAPDH and expressed as fold change relative to mock controls (n=3 biologically independent cells per group). Data are presented as mean ± SEM. (d) At 8 and 14 dpi, mock- or ZIKV-infected human fetal pericytes were subjected to Alizarin red staining for calcium deposition. Data are representative of two independent experiments. (e-f) Alizarin red staining were performed on mock- or ZIKV-infected SK-N-SH and U251 at 14 and 21 dpi. Data are representative of four independent experiments. (g) Human primary fetal pericytes (n=5) and astrocytes (n=6) were infected with PBS (mock) or ZIKV PRVABC59 at MOI of 0.5. At 3 dpi, osteogenic gene expressions were normalized to GAPDH and expressed as fold changes relative to mock controls. Data are presented as mean ± SEM and are representative of two independent experiments. (h) RNAseq data generated from Nature Neuroscience; 2017; 20(9); p1209–1212, doi: 10.1038/nn.4612 (GEO ID: GSE87750) was reanalyzed for osteogenic gene expression. Human primary peripheral neurons were infected with ZIKV PRVABC59 of MOI 0.4 and harvested at 3 dpi. Statistical analyses were performed using one-way ANOVA with Tukey’s posttest (a and c). ***P< 0.001 and ****P< 0.0001. Exact P values in a compared between ZIKV MR766 and ZIKV H/PF/2013 group (P= 0.0008), ZIKV MR766 and ZIKV PRVABC59 group (P= 0.0005), ZIKV IBH30656 and ZIKV H/PF/2013 group (P= <0.0001) and ZIKV IBH30656 and ZIKV PRVABC59 group (P= <0.0001).

Extended Data Fig. 3. ZIKV infection of 3-weeks-old hSTAT2KI mice resulted in brain calcification.

Three-week-old hSTAT2KI were s.c. infected with ZIKV.(a) At 8 dpi, ZIKV RNA was quantified in the brain (MR766 n=7; PRVABC59 n=5). (b) Osteogenic gene expression in mock- or ZIKV-infected hSTAT2KI brains (PBS n=5; MR766 n=6; PRVABC59 n=6) were normalized to GAPDH and presented as fold changes relative to mock controls. (c) Brain sections were stained with H&E or Alizarin red for calcium deposit. Black dotted line in the magnified insert indicates vasculature and blue dotted line in the magnified insert indicates prominent calcification sites. Data are presented as mean ± SEM, using Mann-Whitney U-test (a) or two-way ANOVA followed by Tukey’s multiple comparisons test (b). *P< 0.05, **P< 0.01 and ***P< 0.001. Exact P values in a compared between ZIKV MR766 and ZIKV PRVABC59 group (P= 0.0051). Data in a-c are representative of three independent experiments.

Extended Data Fig. 4. ZIKV infection of osteoblastic-like cells increase osteogenic gene expression.

(a-b) Mock- and ZIKV-infected U2OS cells were harvested at 2 and 4 dpi respectively for osteogenic gene expression. (c) Band intensity of pSMAD1/5/9 from U2OS whole cell lysate. (d) At day 1–4, IgG or nAb-treated and mock or ZIKV-infected U2OS cells (n=6) were harvested for RNA extraction and viral load against ZIKV NS1 RNA was quantified using qRT-PCR (N.D.; Not detected) (e) Human primary fetal brain pericytes are infected with ZIKV PRVABC59 or PBS (mock control) that were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls (n=4/group). Data in a, b, d and e are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons (a), Mann-Whitney U-test (b), or two-tailed unpaired Student t-test (e). *P< 0.05, **P< 0.01, ***P< 0.001. Exact P values in b compared between Mock and ZIKV H/PF/2013 (BMP2 P= 0.0002; RUNX2 P= 0.0011; OSX P= 0.0002; ALPL P= 0.0002; DMP1 P= 0.0002 and PDPN P= 0.0002). Exact P values in e compared between IgG-treated and Nab-treated group (BMP2 P= 0.0054; RUNX2 P= 0.0005; OSX P= 0.0085; DMP1 P= 0.0099 and PDPN P= 0.0005). Data are representative of two independent experiments.

Extended Data Fig. 5. Overexpression of ZIKV NS3 protease induced osteogenic gene expression in osteoblastic-like cells.

U2OS cells (n=6) were transiently transfected with plasmids encoding individual ZIKV genes for 3 days. Transfected cells were harvested for qRT-PCR analysis of osteogenic gene expressions. Gene expressions were normalized to GAPDH and expressed as fold changes relative to vector control. Data are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are representative of three independent experiments.

Extended Data Fig. 6. ZIKV NS3 protease is highly conserved across different lineages.

(a) Sequence alignment comparison between BMP2 gene in human (homo sapiens; amino acid residues 1–396) and hamster (Cricetulus; amino acid residues 1–399). (b) Purity of BL21 strain-derived recombinant ZIKV NS3 protease, CHO-FD11 cells-derived full-length BMP2 and BMP4 were determined by coomassie blue stain. MW: molecular weight. (c) Sequence alignment comparison between human BMP2 (amino acid residues 1–396) and BMP4 (amino acid residues 1–408). (d) Sequence alignment of NS3 protease domain (amino acid residues 1–177) was compared across two African ZIKV (MR766, IbH30656) and two Asian ZIKV (PRVABC59 and H/PF/2103). The red-colored letters indicate protease catalytic triad. (e) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP4 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. Data in b and e are representative of three independent repeats.

Fig. 1. Calcification in brain specimens from ZIKV-infected fetuses.

(a) CT scan of calcifications (red arrows) in the subcortical white matter in a 9-month-old infant infected with ZIKV during second trimester of pregnancy. (b) Brain tissue sections (10μm) from ZIKV-positive human fetuses derived from stillbirths were stained with Von Kossa (top) and H&E (bottom). (c) Serial brain sections from ZIKV-positive human fetal brain tissue were stained with Von Kossa (calcium; black), anti-PDGFRβ (pericytes), anti-GFAP (astrocytes), or anti-NeuN (neurons). Images were taken at 10x magnification (top panel) and 20x magnification (bottom panel) (d) Von Kossa and RNAScope duplex in-situ hybridization with ZIKV RNA and PDGFRβ mRNA (pericytes) in serially sectioned ZIKV-positive human fetal brain tissues. Brightness and contrast were adjusted in magnified insert. Red arrows indicate PDGFRβ+/ ZIKV+ pericytes. The data are representative images from five ZIKV-positive fetal donors.

Fig. 2. Osteogenic gene expression and calcification in ZIKV+ fetal pericytes.

(a) Fetal pericytes (CD146⁺/CD31⁻) were infected with African strain ZIKV-MR766, Asian strains ZIKV-H/PF/2013 and PRVABC59 at MOI of 0.5 or treated with medium without virus (mock). Blue cell population is unstained control, red cell population is stained with CD146 and CD31 antibody. See Supplementary Data Fig. 1 for gating strategy. (b) At 1–4 and 8 dpi, cells were harvested for viral RNA analysis (n=4). (c) Infectious virus titer was quantified in supernatants (n=6 per group) collected from 1–4, 8 and 14 dpi. (d) At 3, 4 and 8 dpi, mature BMP2 protein from supernatants of mock- and ZIKV-infected pericytes was measured (n=8). (e) Osteogenic gene expression in fetal pericytes was normalized to GAPDH and presented as fold changes relative to mock controls (n=6 per group). (f-g) Alizarin red staining and Alizarin red concentration (mM) of ZIKV-infected fetal pericytes after 14 dpi (n=4). Data are presented as mean ± SEM, using two-way ANOVA followed by Sidak’s multiple comparisons test (e) or one-way (g) ANOVA with Tukey’s posttest. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001. Data in d, e and g are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Exact P values in g compared between Mock and ZIKV H/PF/2013 (P= 0.0046) and between Mock and PRVABC59 (P= 0.0030). Data are representative of three independent experiments.

Fig. 3. Vertical transmission of ZIKV induces brain calcification in mice.

(a) Schematic depicting the timeline for our ZIKV-vertical transmission mouse model. Time-mated immunocompetent hSTAT2 KI pregnant dams (8 to 12 weeks old) were i.p infected with PBS (mock control) or ZIKV PRVABC59 at mouse embryonic day 13.5 (E13.5) and pups were born between 19 to 21 days later. On post-delivery day (PD) 28, ZIKV-infected hSTAT2KI pups were sacrificed and brains were harvested for RNA extraction or cryo-sectioning. (b) Osteogenic gene expression in mock- (n =3) or ZIKV-infected hSTAT2KI pup brains (n=12) were normalized to GAPDH and presented as fold changes relative to mock controls. (c) At PD28, brain sections of pups were stained with DAPI (blue) and anti-NS2B antibody. (d) Brain sections were stained with Alizarin red for calcium deposit. Blue arrows indicate the locations of prominent calcium deposition. Data in b-d are representative of two independent experiments. Data in b are presented as mean ± SEM, using two-tailed unpaired Student t-test. *P< 0.05, **P< 0.01 and ****P< 0.0001. Exact P values in b compared between PBS and ZIKV PRVABC59 (Bmp2 P= < 0.0001; Runx2 P= 0.0027; Osx P= 0.0180; Alpl P= < 0.0001).

Fig. 4. ZIKV-induced calcification is dependent on BMP2 signaling.

(A-B) U2OS cells were infected with ZIKV at an MOI of 0.5. (a) Supernatants from mock- and ZIKV-infected cultures were harvested for viral plaque assays (n=8 biologically independent samples per group), and (b) cells were tested for viral RNA (n=4 biologically independent samples per group). Data in b are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. (c) At 1–4 dpi, cell viability was determined by flow cytometry. See Supplementary Data Fig. 1 for gating strategy. (d) At 1–3 dpi, mock- and ZIKV-infected U2OS (MR766: MR; H/PF/2013: HPF) and their supernatants were collected, and TCA precipitated for immunoblot analysis with anti-BMP2 and Ponceau staining. (e) Phosphorylated SMAD1/5/9, total SMAD1 and ZIKV NS3 from whole cell lysates were detected by immunoblotting with indicated antibodies. (f-h) Inhibition of BMP2 activity. Mock- or ZIKV-infected U2OS cells were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. (f) At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls (n=3 biologically independent cells per group). (g) At 21 dpi, cells were stained with Alizarin red stain (calcium) and (h) Alizarin red stain concentration was quantified (n=6). All results are representative of biological independent replicates from two independent experiments. Data are presented as mean ± SEM, using two-tailed unpaired Student t-test (f), two-way ANOVA followed by Sidak’s multiple comparisons test (b), or one-way ANOVA with Tukey’s posttest (h). *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001. Exact P values in f compared between IgG and Nab group (BMP2 P= 0.0034; RUNX2 P= 0.0267; OSX P= 0.0054; DMP1 P= 0.0113 and PDPN P= 0.0105). Significance differences of the means in h comparing between ZIKV-H/PF/2013 +IgG and ZIKV-H/PF/2013 +Nab group (P= <0.0001)

Fig. 5. ZIKV NS3 serine protease upregulates osteogenic signaling in fetal pericytes.

(a) Primary fetal pericytes transduced with lentivirus encoding individual ZIKV protein. Lenti-transduced cells were harvested for analysis of osteogenic gene expression normalized to GAPDH and expressed as fold changes relative to vector control (n=8). (b) Schematic of ZIKV NS3 protease WT and H51A/S135A mutant (ZIKV NS3_Mut). (c) Osteogenic gene expressions in WT and mutant (Mut) NS3-transduced pericytes (n=4). Data in a and c are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are presented as mean ± SEM, using two-tailed unpaired Student t-test. **P< 0.01, ***P< 0.001, ****P< 0.0001. Exact P values in c compared between WT and Mut NS3 group (BMP2 P= 0.0012; RUNX2 P= 0.0013; OSX P= 0.0002; DMP1 P= 0.0030; PDPN P= <0.0001). Data are representative of three independent experiments.

Fig. 6. Purified ZIKV NS3 serine protease processes pro-BMP2 to biologically active mature form to induce osteogenic signaling.

(a) At 3 days after transfection with Flag-tagged WT or mutant NS3, the supernatants and lysates of U2OS cells were subjected to immunoblotting analysis or Ponceau staining. (b) U2OS stably-expressing WT or mutant NS3 were cultured and stained with Alizarin red. (c) Schematic of predicted BMP2 cleavage products. (d) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP2 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. (e) Cleaved BMP2 products from in vitro reaction were added to U2OS cells for 0 or 15 min and cells were harvested for immunoblot analysis. Data are representative of two independent experiments. (f) Schematic model of ZIKV NS3 protease-induced BMP2 maturation and osteogenic gene expression for fetal brain calcification. ZIKV NS3 protease efficiently cleaves pro-BMP2 to generate secreted mature BMP2 that induces its receptor-mediated osteogenic gene expression and calcification.