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

Nature Microbiology volume 6, pages 455–466 (2021)Cite this article

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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.

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Fig. 1: Calcification in brain specimens from ZIKV-infected fetuses.
Fig. 2: Osteogenic gene expression and calcification in ZIKV+ fetal pericytes.
Fig. 3: Vertical transmission of ZIKV induces brain calcification in mice.
Fig. 4: ZIKV-induced calcification is dependent on BMP2 signalling.
Fig. 5: ZIKV NS3 serine protease upregulates osteogenic signalling in fetal pericytes.
Fig. 6: Purified ZIKV NS3 serine protease processes pro-BMP2 to its biologically active mature forms to induce osteogenic signalling.

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Data availability

The gene expression data presented in Extended Data Fig. 2h of this manuscript were reanalysed from RNA-seq data reported by Oh et al.25 and deposited with the Gene Expression Omnibus under accession no. GSE87750. Data supporting the findings of this study are available from the corresponding author upon reasonable request. All RT–qPCR primers and probe sequences used in this study are listed in Supplementary Table 1. Source data are provided with this paper.

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Acknowledgements

This work was partly supported by: grant no. K99DE028573 to W.C.; grant nos. CA200422, CA251275, AI140705, AI140705S, AI140718, AI152190, AI116585, AI116585S, DE023926, DE027888, DE028521 and KGM9942011 to J.U.J.; grant nos. AI069120, AI056154, AI078389 and AI28697 to G.C.; grant nos. AI129534-01, AI298847-01 to K.N.S.; grant nos. AI140718 to J.U.J., G.C. and K.N.S.; the Departamento de Ciência e Tecnologia (DECIT/25000.072811/2016-17) do Ministério da Saúde do Brasil and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior CAPES/ 88887.116627/2016-01 (P.B.); grant nos. R01AR071734 and R01AG058624 to D.E.; and National Institutes of Health/National Institute of Allergy and Infectious Diseases grant nos. U19AI118610 and R21AI129486 to A.G.‐S.

Author information

Authors and Affiliations

  1. Department of Cancer Biology and Global Center for Pathogens Research and Human Health, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Weiqiang Chen, Suan-Sin Foo, Shin-Ae Lee & Jae U. Jung

  2. Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Eunjin Hong, Christine Wu, Wai-Suet Lee & Denis Evseenko

  3. Clinical Research Unit, Fernandes Figueira Institute-FioCruz, Flamengo, Rio De Janeiro, Brazil

    Maria Elisabeth Lopes Moreira

  4. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Adolfo García-Sastre

  5. Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Adolfo García-Sastre

  6. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Adolfo García-Sastre

  7. The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Adolfo García-Sastre

  8. Department of Microbiology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Genhong Cheng

  9. Division of Pediatric Infectious Diseases, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Karin Nielsen-Saines

  10. Laboratório de Pesquisa Clínica em Doenças Febris Agudas, Instituto Nacional de Infectologia Evandro Chagas, FioCruz, Rio de Janeiro, Brazil

    Patrícia Brasil

  11. Department of Pathology, Fernandes Figueira Institute-FioCruz, Rio De Janeiro, Brazil

    Elyzabeth Avvad-Portari

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  1. Weiqiang Chen
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Contributions

W.C. and J.U.J. designed the experiments. W.C. and S.-S.F. performed the experiments and analysed the data. W.C. prepared the figures and wrote the manuscript. E.H., C.W., S.-A.L. and W.-S.L. assisted with the experiments. D.E., M.E.L.M., A.G.-S., K.N.-S., P.B., G.C. and E.A.-P. provided expertise and tools for the study. J.U.J. edited the manuscript and oversaw all study design and data analysis. All other authors read the manuscript and provided comments.

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Correspondence to Jae U. Jung.

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J.U.J. is a scientific adviser of the Vaccine Stabilization Institute, a California corporation. The remaining authors declare no competing interests.

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Peer review information Nature Microbiology thanks Laurent Nguyen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

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 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).

Source data

Extended Data Fig. 3 ZIKV infection of 3-weeks-old human STAT2 knockin 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.

Source data

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.

Source data

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.

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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.

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Chen, W., Foo, SS., Hong, E. et al. Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses. Nat Microbiol 6, 455–466 (2021). https://doi.org/10.1038/s41564-020-00850-3

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